We thank M.W. laboratory members for scientific discussion. This work is supported by AHA [13SDG16920099] to M.W., and by National Heart, Lung, and Blood Institute grants [R01HL121700] to M.W. Images were captured in the Imaging Core Facility at the Albany Medical College.

全ての動物実験は、アルバニー医科大学で制度動物実験委員会（IACUC）によって承認され、実験動物の管理と使用に関するNIHガイドに従って行いました。 1.ソリューションの準備注：Rosa26Cre ERT2 15、R26R-紙ふぶき 16、αMHC-Creを17、 とのRosa26-mTmG（mTmG）20マウスのラインが商業的に購入したcTnTの-Creを 18は、アラバマ大学の博士交通からの贈り物だったのNFATc1-Creを 。医学19のアルバートアインシュタイン大学の博士ビン周からの贈り物でした。マウスは、二国間の開胸、断頭または頸椎脱臼により死亡検証の物理的手段に続いて、少なくとも60秒間密閉チャンバーに二酸化炭素吸入によって安楽死させました。 <ol><li>タモキシフェンを準備します。ヒマワリ種子大井の10ミリリットルにタモキシフェン10mgを溶かしリットル。 -20℃で完全に溶解したら、アリコートとストア。 </li><li>リン酸緩衝生理食塩水（PBS）を準備します。 Na 2 HPO 4（1.41960グラム）、KH 2 PO 4（0.24496グラム）、塩化ナトリウム（8.0669グラム）及び蒸留水1L中のKCl（0.20129グラム）を溶解し、pHを7.4に調整します。 </li><li> PBSTを調製0.1％のTween-20溶液をPBS中でPBS 100mlのTween-20を100μlの溶解させることによって。 </li><li>ブロッキングバッファーを準備します。 0.2％のTween-20を含有するPBST 100mlにBSAを3gを溶解し、3％ウシ血清アルブミン（BSA）ブロッキング溶液を作ります。 </li><li> CUBIC-1（試薬1）を準備します。 25重量％の尿素、25重量％のN、N、N &#39;、N&#39;-テトラキス（2-ヒドロキシプロピル）エチレンジアミンとのdH 2 O中15重量％のTriton X-100を混合</li><li> CUBIC-2（試薬2）を準備します。 50重量％のショ糖、25重量％の尿素、10重量％の2,2 &#39;、2&#39; &#39;ミックス- nitrilotriethanolおよび0.1％（Vし/ V）のdH 2 O中のトリトンX-100 </li><li> Sca ル A2を準備します。4 M尿素、w / vグリセロール10％および0.1％のw / vのトリトンX-10dH 2 O中0 7.7にpHを調整します。 </li><li> Sca ルの B4を準備しますのdH 2 Oで8 M尿素およびワット/ vのトリトンX-100を0.1％ 8.7にpHを調整します。 </li><li> 4 M尿素、70％のw / vのグリセロールおよび0.1％W / VトリトンX-100 の dH 2 O中：のSca ル 70を準備します</li></ol> 2.タモキシフェン誘導、胚の単離と固定<ol><li>タモキシフェン誘導および胚の単離<ol><li> （R26Cre ERT2雄マウスで塞が）女性R26Cre ERT2紙吹雪マウスは、胚の発達日E7.75 33でタモキシフェンを10μg/ gの湾曲した栄養管を使用して、承認されたプロトコル（ACUP 13から12007）、強制飼養。胎生9.5（E9.5）またはE10.5では、CO 2および頸椎脱臼と雌マウスを安楽死させます。 </li><li>マウスの死亡確認後（セクション1を参照してください。注）、はさみでマウス腹腔を開き、子宮角を解剖、ハサミやピンセットの助けを借りて卵管層を除去し、再子宮管23,24から胚をリース。 <ol><li> PBS（pHは7.4）を含有するペトリ皿に胚を移します。解剖顕微鏡下では、ピンセットの助けを借りて、非胚層（絨毛膜、卵黄嚢と羊膜）を削除。 </li></ol></li><li>以前に22を報告したようにピンセットやハサミを使用して、遺伝子型決定のための雌マウスと胚の尾のサンプルを収集します。プラスチックピペットを用いて、1×PBSの1ミリリットルを含む48ウェルプレートのウェルに各胚を移します。 </li><li>蛍光顕微鏡下では、カメラ付き倒立顕微鏡を介して、GFP / RFP / CFP陽性の胚を識別します。ピール離れてピンセットを用いて、心膜とGFP / RFP / CFPフィルターと心の両面上の蛍光クローンの数を決定するために、湾曲したピンセットの助けを借りて心/胚を回転させます。いいえカットは必要ありません。 </li></ol></li><li> GFP / RFP / CFP陽性の胚を識別した後、すぐに目で1×PBSで2回（各2分）の胚を洗います室温（RT）でプレートシェーカー上で電子48ウェルプレート。これは、過剰な血液を除去します。 </li><li> RTで4％パラホルムアルデヒド（PFA）を使用して、胚/胎児心を修正しました。定着時間は、胚の年齢や組織のサイズによって異なります。 E9.5室温で2時間の胚を修正。後期胚の段階の胚でも生後心臓のために24時間まで延長することができ、より長い定着時間を必要とします。 注意：パラホルムアルデヒドは毒性があり、皮膚、目や粘膜との接触を避けます。 </li><li>固定後、室温でプレートシェーカー上で二回（各10分）1×PBSで48ウェルプレートに胚/心を洗います。これは、胚/心臓から過剰PFAを削除します。胚はその後さらにホールマウント染色および組織をクリアするために処理されます。 </li></ol> 3.ホールマウント染色注：次のようにPFA固定後胚/心臓が全体のマウント染色に供されます。 <ol><li> 1メートルを使用して、48ウェルプレートに胚/心を透過性RTでプレートシェーカー上で2時間、0.1％PBSTのリットル。 </li><li>透過処理に続いて、4ºCでプレートシェーカー上で2時間または一晩、ブロッキング緩衝液1mlに胚/心を浸します。 </li><li> 4ºCでプレートシェーカー上で48時間、ブロッキング緩衝液0.3ミリリットルで：（100 1）で希釈し、内皮細胞特異的抗体PECAM（CD31）に胚/心を浸し。 </li><li>プレートシェーカー上で室温でPBSTを5回、30分ごとを使用して胚/心を洗ってください。これは、組織からの過剰非特異的結合した一次抗体を除去します。 </li><li> 4ºCでプレートシェーカー上で48時間、ブロッキング緩衝液1ミリリットル中：（千1）で希釈し、アレクサフルオロ647ヤギ抗ラット二次抗体で胚/心を浸します。 </li><li>二次抗体を洗浄する手順を繰り返し3.4。 </li><li>プレートシェーカー上で室温で1時間、4％PFAで胚/心を後固定し、PBSで5分間、それぞれで二回洗浄します。 </li><li>コンセントでPBSTで希釈した核染色4 &#39;、6-ジアミジノ-2-フェニルインドール（DAPI）（と胚/心を染色RTでプレートシェーカー上で10分間、0.01 mg / mlで）の比。それぞれ5分の2回のPBS洗浄に従ってください。 </li></ol> 4.胚/のSCA リットルの eまたはキュービック法とハートのクリア注：ホールマウント染色した後、胚/心臓がのSca ルまたはCUBIC組織のクリア方法のいずれかに供されます。方法の選択は、組織のサイズおよび胚の年齢に依存します。 E11.5以前の年齢の胚のための、のSCA リットル電子組織のクリア方法を使用して、古い胚または出生後の心のためにCUBIC組織クリア方法が好ましいが。 <ol><li>規模組織のクリア方法<ol><li>時折穏やかに4℃で48時間（手で）振とうしながら（アルミホイルに包んで）シンチレーションバイアル中のSCA リットル電子 A2溶液1mlと胚/心をインキュベートします。 </li><li> Sca リットル電子のA2のインキュベーションの後、時折穏やかanot振とう同じバイアル中で4℃でのSCA リットル電子B4の溶液1 mLで胚/心をインキュベート彼女の48時間。 </li><li>時折穏やかに振とうしながら4℃で1ミリリットルのSCA リットル電子 70と別の48時間胚/心をインキュベートします。 90％グリセロールを使用してマウント胚（セクション5.1を参照）。 </li></ol></li><li> CUBIC組織のクリア方法<ol><li>全胚/心CUBICクリアするため、時折穏やかに37ºC7で48時間振とうCUBIC-1の1ミリリットル中の各胚/心を浸します。 48時間後、新鮮なCUBIC試薬1の同じ体積で溶液を交換し、時折手で振盪しながら37℃でさらに48時間、サンプルをインキュベートします。 </li><li>穏やかに手で振盪しながら室温で1×PBSで数回CUBIC-1処置した心臓/胚を洗ってください。これは、過剰CUBIC-1溶液を除去します。 </li><li> PBSでCUBIC-1を洗浄した後、時折穏やかに手で振盪しながら37ºCで48時間CUBIC-2溶液（胚/心あたり1グラム）で胚/心を浸します。これは、グリセロールを用いた実装胚/心を次の（第5節参照します0.2）。 </li></ol></li></ol> 5.サンプル取付け・イメージング注：のSCA リットル電子 70またはCUBIC-2溶液中でクリア胚または器官がそれぞれ、イメージング媒体としてのスケール70とCUBIC-2溶液を用いて直接画像化することができます。しかし、試薬をクリアする胚サンプルの脆弱性を考慮すると、サンプルはすぐに画像化されたが、長期的に保存されていない場合、以下のプロトコルに従って、90％グリセロール中でサンプルを格納。 <ol><li>直接のSca LEが撮像されるまで4℃で90％グリセロール、店舗の1mlのサンプルをクリア浸します。 </li><li>二回1×PBSで5分間ずつCUBIC処理サンプルを洗浄し、順次30分間、30％、50％、70％、90％グリセロール溶液をそれぞれ1 mlのサンプルをインキュベートします。 </li><li>イメージングのために、2光子機能を備えた共焦点顕微鏡を用いて、90％のグリセロールおよび画像クローンの最小量のガラス底ペトリ皿に試料を移します。 </li><li> 10X / 0.3 NA、25X / 0.8 NAの浸漬、または40X / 1.2 NA水矯正対物レンズと画像グリセロールで心臓や胚を。サファイアカメレオンレーザー：コヒーレントチタンから2光子励起を用いた画像DAPI。顕微鏡対物レンズの選択は光学シート蛍光顕微鏡を利用することができ、超解像のために、例えば 、実験の目的に依存するであろう。 </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+of+the+mammalian+cardiac+neural.">Fate of the mammalian cardiac neural.</a> Developement. 127, 1607-1616 (2000).</li><li>Viragh, S., Challice, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+of+the+epicardium+and+the+embryonic+myocardial+circulation+in+the+mouse.">The origin of the epicardium and the embryonic myocardial circulation in the mouse.</a> Anat Rec. 201, 157-168 (1981).</li><li>Wu, M., Li, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Numb+family+proteins:+novel+players+in+cardiac+morphogenesis+and+cardiac+progenitor+cell+differentiation.">Numb family proteins: novel players in cardiac morphogenesis and cardiac progenitor cell differentiation.</a> Biomolecular concepts. 6, 137-148 (2015).</li><li>Li, 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=Single-Cell+Lineage+Tracing+Reveals+that+Oriented+Cell+Division+Contributes+to+Trabecular+Morphogenesis+and+Regional+Specification.">Single-Cell Lineage Tracing Reveals that Oriented Cell Division Contributes to Trabecular Morphogenesis and Regional Specification.</a> Cell reports. 15, 158-170 (2016).</li><li>Alkaabi, K. M., Yafea, A., Ashraf, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+pH+on+thermal-+and+chemical-induced+denaturation+of+GFP.">Effect of pH on thermal- and chemical-induced denaturation of GFP.</a> Appl Biochem Biotechnol. 126, 149-156 (2005).</li><li>Captur, G., 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=Morphogenesis+of+myocardial+trabeculae+in+the+mouse+embryo.">Morphogenesis of myocardial trabeculae in the mouse embryo.</a> J Anat. , (2016).</li></ol>

The authors declare no competing financial interests.

胚の分離は非常に重要なステップです。 E9.5胚ので細心の注意が単離中の胚/心臓の構造に損傷を与えないように注意する必要があり、サイズは非常に壊れやすいと小さいです。胚全体を撮像する場合、胚/心臓を包む非胚余分な層は、特に慎重に除去しなければなりません。これは、胚組織の奥深く抗体および清算混合物の浸透を可能にし、また、撮像する際のバックグラウンド信号を除去す...

心臓の形態形成は、心臓の別個のセクタに心臓前駆細胞の4つの異なるタイプの時空間組織を必要とし、また、機能的な心1,2を形成するために、このプロセスを調整するために、複数の遺伝子制御ネットワークを必要とするシーケンシャルイベントです。心臓の仕様、分化、パターニング、及びチャンバ成熟は心原性転写因子3によって規制されています。心臓前駆細胞の遺伝的変異またはこれらの因子の転写後収差は、胚性致死または先天性心臓欠陥（CHD）4のいずれかになる可能性があります。心臓の形態形成の研究では、3次元（3D）に固有の構造的な詳細を理解する必要がありますし、心臓の開発中にトレース単一ラベルされた心臓前駆細胞系譜は、心臓の形態形成の理解を促進します。高解像度の部分ベース断層撮影法の数が目に開発されています画像臓器構造5,6に数十年の過去の電子。しかしながら、これらの方法は高価で特殊な機器、広範な労働力を必要とし、最終的な体積再構成された画像7,8における単細胞解像度で詳細な構造組織を欠いています。 単一細胞レベルでの3Dボリュームイメージングは、 生体 7 に前駆細胞の分化および細胞の挙動を研究するための手段を提供します。しかし、組織の光散乱が深く、無傷の心の中の3Dでの細胞や構造を画像化する主要な障害となっています。脂質は、光散乱の主要な供給源であり、脂質および/または脂質およびその周辺地域との間の屈折率差の調整の除去は、組織透明8を高めるための潜在的なアプローチです。過去数年間で、組織のクリア方法の数は、組織の不透明度と光の散乱を低減しており、開発されたBABB（ベンジルアルコール、ベンジルbenzoatなどEの混合物）とDBE（テトラヒドロフラン、ジベンジル）;しかし、これらの方法では、蛍光消光が問題8-10まま。溶媒としては、例えばSeeDB（フルクトース/チオグリセロール）と3DISCO（ジクロロメタン/ジベンジル）などの親水性の方法を、ベースの蛍光シグナルを維持するが、7,8,11透明な臓器全体をレンダリングしません。比較では、CLARITY組織クリア方法は、透明な臓器をレンダリングするが、それはまた、7,13を埋め込むヒドロゲルを必要とする、PACT（パッシブ明確にする技術が）同じように、脂質8,12を除去するために、特殊な電気泳動装置を必要とします。利用可能なすべての組織クリアする方法に関する詳細情報については、 ら 、リチャードソンとリヒトマンの表1を参照してください。7。 2011年に、ハマらは偶然マウス脳と胚transparをレンダリングする親水性混合物」のSca ル 」（尿素、グリセロールおよびTriton X-100混合物）を発見しましたENT完全に標識されたクローン14からの蛍光シグナルを維持しながら。これは、無傷の数ミリメートルの深さで脳や細胞内の解像度でニューロン集団と突起の大規模な再構成の撮像を可能にします。須崎ら。さらにクリア時間を減少させ、リン脂質可溶化を増加し、多色蛍光イメージング8に許可された組織のクリア方法は、アミノアルコールを添加することにより、のSca ルを改善し、「CUBIC」（クリア、遮るもののない脳画像カクテルやコンピュータ解析）を開発しました。本研究では、心臓の間に心の中のSca ルの利点とCUBIC組織決済技術と高解像度の3D光学切片、個々のクローンを取ることはRosa26Cre ERT2 15、R26R-紙ふぶき 16、αMHC-Creを17、cTnTの-のCreを使用して追跡しました18、 </strong&gt; NFATC1-Creを 19、とのRosa26-mTmG（mTmG）20マウス系統。ホールマウント染色（WMS）メソッドの組み合わせは、以前に21,22、さらに標識されたクローン中の他のタンパク質の染色のためにと3Dボリュームコンテキストで彼らの行動の研究のために許可された組織のクリア方法とを開発しました。組織のクリア及びWMSの組合せは、心臓の開発中の異なる遺伝子およびタンパク質の役割のよりよい理解を可能にし、先天性心臓欠陥の病因。このプロトコルは、開発中に、他の前駆細胞の分化、細胞の挙動、および臓器形態形成事象を研究するために適用することができます。

<table border="0" cellpadding="0" cellspacing="0" width="950">
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">2,2&#8242;,2&#8242;&#8217;-nitrilotriethanol&#160;</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">90279</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4% Paraformaldehyde in PBS</td>
			<td style="width:192px;">Affymetrix</td>
			<td>19943</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA</td>
			<td style="width:192px;">Fischer Scientific&#160;</td>
			<td style="width:211px;">BP16000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">N,N,N&#8217;,N&#8217;-tetrakis(2-hydroxypropyl) ethylenediamine&#160;</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">122262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate Buffer Saline</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">P5368-10PAK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton&#160;X-100</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td>T8787</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Urea</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">U-1250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Sucrose</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">84097</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Glycerol</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">G8773</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Tamoxifen</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">T5648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Sunflower seed oil</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">S5007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">P1379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">PECAM (CD31)</td>
			<td>BD Pharmingen&#160;</td>
			<td style="width:211px;">550274</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 647&#160;</td>
			<td>Invitrogen</td>
			<td>A-21247</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">DAPI nuclear stain</td>
			<td style="width:192px;">Sigma Aldrich</td>
			<td style="width:211px;">D9542</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">37oC Incubator</td>
			<td style="width:192px;">Thermoscientific Fischer</td>
			<td style="width:211px;">Heratherm, Compact Microbiological Incubators</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">48 well plates</td>
			<td style="width:192px;">Cell Treat</td>
			<td style="width:211px;">229148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Analytical Balance</td>
			<td style="width:192px;">Metler Toledo</td>
			<td>PB153-S/FACT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Confocal microscope</td>
			<td>Zeiss</td>
			<td>Zeiss 510 confocal microscope</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Disecting Microscope</td>
			<td style="width:192px;">Unitron</td>
			<td style="width:211px;">Z850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescent microscope</td>
			<td>Zeiss</td>
			<td>Observer. Z1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Germinator 500 Glass Bead Sterilizer</td>
			<td style="width:192px;">CellPoint Scientific</td>
			<td style="width:211px;">GER-5287-120V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Light Source</td>
			<td style="width:192px;">SCHOTT</td>
			<td style="width:211px;">ACE I</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pair of Scissors</td>
			<td>Fine Science Tools</td>
			<td>14084-08</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri dish 60x15mm&#160;</td>
			<td align="left">TPP Techno Plastic Products AG</td>
			<td>93060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:381px;">Rocker II&#160; Platform Rocker</td>
			<td>Boekel Scientific</td>
			<td style="width:211px;">260350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scintillating tubes</td>
			<td style="width:192px;">Fischer Scientific&#160;</td>
			<td>03-337-26</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transfer pipette</td>
			<td style="width:192px;">Samco Scientific</td>
			<td style="width:211px;">202</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tweezers</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

imaging cleared embryonic and postnatal hearts at single-cell resolution

全心臓レベルで単一のクローン追跡および分析は、心臓の開発中に、心臓前駆細胞の挙動および分化を決定し、正常と異常な心臓の形態形成の細胞および分子基盤の研究を可能にすることができます。レトロスペクティブ単一クローン解析の最近の新技術は、実現可能な単一細胞の解像度で心臓の形態形成の研究を行います。しかし、撮像深さ、心臓の組織の不透明性と光散乱は単細胞解像度で妨げる全心臓イメージングを増加させます。これらの障害を克服するために、照明および検出の両方のための透明性の高い心をレンダリングすることができます全胚清算システムが開発されなければなりません。幸いなことに、ここ数年では、このようなCLARITYなどの全生物の決済システム、のSca ル 、SeeDB、ClearT、3DISCO、CUBIC、DBE、BABBとPACTのための多くの方法が報告されています。このラボでは、カードの細胞および分子メカニズムに興味がありますIACの形態形成。最近、我々はROSA26-CreをERT2を経由してトレース単一細胞系統を確立し、ROSA26-紙吹雪システムまばらにラベル細胞に心臓の開発中に。私たちは、心の内の画像の単一クローンにホールマウント染色と組み合わせて胚をクリアするためのSca ルとCUBIC（クリア、遮るもののない脳画像カクテルやコンピュータ解析）を含むいくつかの胚全体-クリアする方法論を適応しました。心臓が正常に単一細胞の解像度で画像化しました。私たちは、SCA ルは、胚の心をクリアすることができることを発見しましたが、組織を効果的に溶解することによって胚の心をCUBICは、生後の心をクリアすることができますが、生後の心をクリアしますが、損害賠償はできません。ここに記載される方法は、順番に、先天性心臓欠陥の細胞および分子基盤を明らかにすることができ、心臓の形態形成、間に単一クローンの解像度での遺伝子機能の研究を可能にします。

クリア胚の心臓を画像化脊椎動物の心臓の形成は時空間的に調節形態形成のプロセスであり、4つの異なるソース1からの組織および前駆細胞の分化に依存します。心臓三日月の最初の心臓領域からの細胞は、線形心管を形成するために、腹側正中線に向かって折ります。最初に最初の心臓領域にdorsomediallyに存在す?...

Watch this Scientific Journal Video about Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution at JoVE.com

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

イメージングは​​、単一細胞の解像度で胚および出生後の心をクリア

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

imaging-cleared-embryonic-postnatal-hearts-at-single-cell

Research

JoVE Journal

Developmental Biology

8.1K ビュー.  Albany Medical College. We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately. 

ビデオ: Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

内イメージングカルシウム<em&gt;ショウジョウバエ</em&gt;卵活性化で

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

発生生物学

Reprogramming Mouse Embryonic Fibroblasts with Transcription Factors to Induce a Hemogenic Program

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We first designed a reporter screen using MEFs from human CD34-tTA/TetO-H2BGFP (34/H2BGFP) double transgenic mice. CD34+ cells from these mice label H2B histones with GFP, and cease labeling upon addition of doxycycline (DOX). MEFS were transduced with candidate TFs and then observed for the emergence of GFP+ cells that would indicate the acquisition of a hematopoietic or endothelial cell fate. Starting with 18 candidate TFs, and through a process of combinatorial elimination, we obtained a minimal set of factors that would induce the highest percentage of GFP+ cells. We found that Gata2, Gfi1b, and cFos were necessary and the addition of Etv6 provided the optimal induction. A series of gene expression analyses done at different time points during the reprogramming process revealed that these cells appeared to go through a precursor cell that underwent an endothelial to hematopoietic transition (EHT). As such, this reprogramming process mimics developmental hematopoiesis "in a dish," allowing study of hematopoiesis in vitro and a platform to identify the mechanisms that underlie this specification. This methodology also provides a framework for translation of this work to the human system in the hopes of generating an alternative source of patient-specific hematopoietic stem cells (HSCs) for a number of applications in the treatment and study of hematologic diseases.

The protocol described here details the induction of a hemogenic program in mouse embryonic fibroblasts via overexpression of a minimal set of transcription factors. This technology may be translated to the human system to provide platforms for future study of hematopoiesis, hematologic disease, and hematopoietic stem cell transplant.

転写因子との再プログラミングマウス胚線維芽細胞造血プログラムを誘導するために

This protocol details the induction of a hemogenic program in mouse embryonic fibroblasts (MEFs) via overexpression of transcription factors (TFs). We ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

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

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

クリア胚ハーツにおける冠状血管の分析

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

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.

Light-mediated Reversible Modulation of the Mitogen-activated Protein Kinase Pathway during Cell Differentiation and Xenopus Embryonic Development

This protocol describes an optogenetic strategy to modulate mitogen-activated protein kinase (MAPK) activity during cell differentiation and Xenopus embryonic development. This method allows for the reversible activation of the MAPK signaling pathway in mammalian cell culture and in multicellular live organisms, like Xenopus embryos, with high spatial and temporal resolution.

細胞分化中のマイトジェン活性化プロテインキナーゼ経路の光媒介可逆的調節<em&gt;アフリカツメガエル</em&gt;胚発生

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early ...

Isotropic Light-Sheet Microscopy and Automated Cell Lineage Analyses to Catalogue Caenorhabditis elegans Embryogenesis with Subcellular Resolution

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous system can be observed, with single cell resolution, in vivo. Here, we present an integrated protocol for the examination of neurodevelopment in C. elegans embryos. Our protocol combines imaging, lineaging and neuroanatomical tracing of single cells in developing embryos. We achieve long-term, four-dimensional (4D) imaging of living C. elegans&#160;embryos with nearly isotropic spatial resolution through the use of Dual-view Inverted Selective Plane Illumination Microscopy (diSPIM). Nuclei and neuronal structures in the nematode embryos are imaged and isotropically fused to yield images with resolution of ~330 nm in all three dimensions. These minute-by-minute high-resolution 4D data sets are then analyzed to correlate definitive cell-lineage identities with gene expression and morphological dynamics at single-cell and subcellular levels of detail. Our protocol is structured to enable modular implementation of each of the described steps and enhance studies on embryogenesis, gene expression, or neurodevelopment.

Here, we present a combinatorial approach using high-resolution microscopy, computational tools, and single-cell labeling in living C. elegans embryos to understand single cell dynamics during neurodevelopment.

等方性光シート顕微鏡と自動細胞系譜分析による、細胞内分解能を Caenorhabditis elegans 胚発生

Caenorhabditis elegans (C. elegans) stands out as the only organism in which the challenge of understanding the cellular origins of an entire nervous ...

Capturing the Cardiac Injury Response of Targeted Cell Populations via Cleared Heart Three-Dimensional Imaging

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in cardiac injury, primarily in the form of an acute myocardial infarction. With little resilience following injury, the once healthy cardiac tissue will be replaced by fibrous, non-contractile scar tissue and often be a prelude to heart failure. To identify novel treatment options in regenerative medicine, research has focused on vertebrates with innate regenerative capabilities. One such model organism is the neonatal mouse, which responds to cardiac injury with robust myocardial regeneration. In order to induce an injury in the neonatal mouse that is clinically relevant, we have developed a surgery to occlude the left anterior descending artery (LAD), mirroring a myocardial infarction triggered by atherosclerosis in the human heart. When matched with the technology to track changes both within cardiomyocytes and non-myocyte populations, this model provides us with a platform to identify the mechanisms that guide heart regeneration. Gaining insight into changes in cardiac cell populations following injury once relied heavily on methods such as tissue sectioning and histological examination, which are limited to two-dimensional analysis and often damage the tissue in the process. Moreover, these methods lack the ability to trace changes in cell lineages, instead providing merely a snapshot of the injury response. Here, we describe how technologically advanced methods in lineage tracing models, whole organ clearing, and three-dimensional (3D) whole-mount microscopy can be used to elucidate mechanisms of cardiac repair. With our protocol for neonatal mouse myocardial infarction surgery, tissue clearing, and 3D whole organ imaging, the complex pathways that induce cardiomyocyte proliferation can be unraveled, revealing novel therapeutic targets for cardiac regeneration.

Cardiomyocyte proliferation following injury is a dynamic process that requires a symphony of extracellular cues from non-myocyte cell populations. Utilizing lineage tracing, passive CLARITY, and three-dimensional whole-mount confocal microscopy techniques, we can analyze the influence of a variety of cell types on cardiac repair and regeneration.

クリアされた心臓3次元イメージングによる標的細胞集団の心臓損傷応答のキャプチャ

Cardiovascular disease outranks all other causes of death and is responsible for a staggering 31% of mortalities worldwide. This disease manifests in ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

遺伝子発現解析のためのマウス胚軟骨と骨のレーザー捕捉微小分片

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

ヒト胚性幹細胞の単細胞培養を用いた効率的な神経分化

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

マウス胚細胞の多重化単細胞mRNAシーケンシング解析

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Vascular Casting of Adult and Early Postnatal Mouse Lungs for Micro-CT Imaging

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and behave by observing the surface of a tissue. This method provides a means to visualize the complex 3-dimensional vascular architecture of the lung.
To accomplish this, a catheter is inserted into the pulmonary artery and the vasculature is simultaneously flushed of blood and chemically dilated to limit resistance. Lungs are then inflated through the trachea at a standard pressure and the polymer compound is infused into the vascular bed at a standard flow rate. Once the entire arterial network is filled and allowed to cure, the lung vasculature may be visualized directly or imaged on a micro-CT (&#181;CT) scanner.
When performed successfully, one can appreciate the pulmonary arterial network in mice ranging from early postnatal ages to adults. Additionally, while demonstrated in the pulmonary arterial bed, this method can be applied to any vascular bed with optimized catheter placement and endpoints.

The aim of this technique is ex vivo visualization of pulmonary arterial networks of early postnatal and adult mice through lung inflation and injection of a radio-opaque polymer-based compound via the pulmonary artery. Potential applications for casted tissues are also discussed.

マイクロCTイメージング用成人および早期産後マウス肺の血管鋳造

Blood vessels form intricate networks in 3-dimensional space. Consequently, it is difficult to visually appreciate how vascular networks interact and ...

イメージングは​​、単一細胞の解像度で胚および出生後の心をクリア

イメージングは、単一細胞の解像度で胚および出生後の心をクリア