The authors would like to thank Dr. George Gittes for advice and expertise throughout this study. SSL was supported by an American Heart Association fellowship (11POST7330002). Further to this SSL and this study was supported by an NIDDK Mentored Research Scientist Development Award (DK096996) and by the Children&#8217;s Hospital of Pittsburgh.

注：ピッツバーグ大学の施設内動物管理使用委員会は、すべての実験を承認した。 超音波マイクロインジェクションインスツルメンツおよび胚の作製<ol><li>ステージを設定し、マウントして、プローブ（ 図1）と同様に手術器具（ 図2）。 37℃の加温槽内の場所のリン酸緩衝生理食塩水（PBS）溶液（pH7.4）で。そのベースを介して、第二の柔軟な25 G針に付着した1mlシリンジを使用して、鉱油を完全にマイクロインジェクション針を埋める。 </li><li>回転上に針を修正アームをマウントし、鉱油溶液の空の針。 TL溶液を2.5μlの再埋める。気泡が注射針内に存在しないことを確認してください。離れてステージから、壁に向かって針アームを回転させます。 </li><li>イソフルランの連続注入を介して麻酔室で妊娠中の母親を麻酔。母親が無意識にレンダリングされると、C上に位置する鼻チューブに麻酔を転送audalステージの側面、そして完全に麻酔をかけた状態の継続を可能にするために鼻のチューブに鼻と仰臥位で母親を置く。 </li><li> 45°の角度で、または手や足にテープ手足は休息と頭上ECG /温度モニタのタブを配置。これは、妊娠中のダムが継続的にその麻酔を確実に監視することが重要であるのに十分であり、眼への印加中にその軟膏は、乾燥を軽減する。 </li><li>優しく余分な製品と毛を除去するために70％エタノール飽和綿棒で再度乾燥した綿棒で拭き取っ、そして、母親の下腹部全体に脱毛製品を適用します。切開部位（ 図3）で、すべての髪のオフ腹部の皮膚をきれいにすることを確認してください。 </li><li>細かい外科鉗子やハサミを使って妊娠中の母親に開腹を行います。すべての主要な血管や内臓器官を切断しないように注意してください。 1.5〜2センチメートル膣の上に、第一表皮のストレート切開を作成し、approximatためのリブに向けてカットを継続エリー2-3センチ。内臓器官および子宮球形嚢の内部を露出させるために、同じ長さに沿って、皮下膜を公開白線（「白線」）（ 図3）を探して、最初の切開に平行に切断。 </li></ol>胚の2.抽出<ol><li>母と胚を操縦するために6インチの綿棒アプリケーターを使用してください。静かに切開口から最初の胚を操作するアプリケータを用いて皮膚に（強制しない）押してください。最初の胚球形嚢の抽出後、静かにゆっくりと通過して、母親の外側に子宮角の残りの部分を引っ張る。この段階で切開を通って腸または他の臓器を引っ張っては避けてください。 </li><li>胚を定量化し、優しく母親に戻す左子宮角に配置します。そして、ホーンに膣から胚球形嚢遠いから始まって下方向に移動バック母に右子宮角を配置し始める。唯一の2胚を露出されたままになるまでこれを続けます（FIグレ4）。最後に、切開線に上記と平行の列の位置の胚は、子宮の循環を遮断しないように認識している。 </li><li>粘土ブロックと腕と体の間の位置に配置し、だけでなく、足や尾。粘土表面は開腹切開とほぼレベルであることを確認してください。 37℃のPBSで有窓ペトリ皿を湿ら。 </li><li>左手（胚に開窓パラレル）で右手と有窓シャーレに鉗子を拡張つかみ、ペトリ皿の上から、窓を通して〜5cmのクローズ鉗子を持って来る。完全に開窓メッシュを引き裂くことなくオープン鉗子。 </li><li>胚の上に手を操縦し、2胚球形嚢に視力を集中。なし（または軽く）スリットを介してそれらをスライドさせ、母親の腹部上にシャーレを設定し、開窓メッシングまたは鉗子で球形嚢に触れる。鉗子はまだ延長すると、いずれかの側に、それぞれの胚のベース（ 図5でそれを着座させる有窓メッシュを操作する</st栄&gt;）と、スリット開口部から鉗子を引き出します。 </li><li>次に、（ 図6）つまんまたは胚を傷つけることなく、シャーレに壁を含む青いゴムを配置します。確認粘土ブロックはしっかりとペトリ皿、胚、およびゴム含有壁をバランスされていることを確認。有窓メッシングで漏れを制御しながら、胚が完全に、（ 図6）水没するまで最後に、37℃のPBSでペトリ皿を埋める。 </li></ol> 3.注入手順<ol><li>次に、下部注入母胚ダウンZレベル調整つまみ（ 図1）を使用して、ステージとプローブ先端左と下針1/2センチで、注射針を回転させ、アームをマウントし、超音波プローブに直接並ぶ（ 図7） 。 45°離れたプローブからの針アーム（しない針）を押します。皿または超音波プローブを用いて針の先端をヒットしないように注意してください。 </li><li>超音波pで、元の高さに噴射ステージを上げる直接プローブがわずかに沈めなければならない胚上方および胚組織3-4 mm以内ローブ。使用X＆Yステージ調整ノブ（ 図8）体系的に破って、胚の心を見つけるための胚/母​​/ステージ位置を修正する。 </li><li>直接超音波画面の中央には、描かれた黒、センター標的マーカー（*）を観察する。 （ 図8）X＆Yステージ調整ノブを使用して、左心室（望ましい）またはアトリウムを見つけて、超音波画面上の黒いセンターマーカーの上に正確に注入ターゲットを配置するために、ステージ上の回転ノブを使用して、ステージを上げるか、下げる。 </li><li>超音波画面から、右に胚を移動するXステージ調整ノブを使用してください。 1/2離れてからCMと、超音波プローブに90°（ 図7）、マイクロインジェクション針の位置を変更し、元の位置に戻し腕。 </li><li>マイクロインジェクションを使用すると、アル明らかに調整つまみ（ 図9C-G）、先端を位置の針をマウントセンターマーカーでigned。針の先端が正しい平面内に集束されるように（ 図9CおよびEGでノブを使用して）噴射角を調整し、マイクロニードルのX、Y、およびZベクター。他の寸法を調整しないように注意し、単に「注射」ノブ（ 図9F）を使用して、マイクロインジェクション針を撤回。 </li><li>ここでも、正確に超音波画面上の中心マークのみにターゲットを持つバックプローブ下のX微調整段階つまみ移動胚を、使用して。左心室が全く同じX、Y、Zの平面上になっていることを確認してください。 </li><li>次に、マイクロインジェクションに単に「注射」ノブでマウント、ゆっくりとマイクロインジェクション針で胚を穿刺し、徐々に左心室に先端をもたらす。 「空」を保持し、（ 図2A＆B）いったん「埋める」をタップして、左心室にまたは空の警告ビープ音がするまで、TL·ソリューションの完全な2.5μLを注入。現時点では注射の心臓の室に入るTL（ 図10）である針の先端から発せられる影を観察する。逆に、すぐに注入が完了したときにマイクロインジェクションの「注射」ノブ（ 図9F）がマウント回転マイクロインジェクション針を撤回。 </li><li>次の胚に進みとこの一連の以下の残りの胚について、この手順を繰り返し、最終的に戻ってダムの腹部への最終胚を置きます。チャンバーボックスに麻酔を切り替えて、静かに最後の注射の時から15分間、ここで母を移動します。 </li></ol> 4.収穫胚および分析<ol><li>頸椎脱臼を経由してダムを生け贄に捧げる。簡単に母親から子宮角を除去するための開腹切開を展開します。子宮嚢の中の胚にしてください。チルドPBS中で胚を置きます。 </li><li> （あれば遺伝子型判定のために必要な収集組織）子宮嚢から胚を解剖し、全胚を置くI一晩30％ショ糖に一晩、その後nは4％パラホルムアルデヒド（PFA）、クライオスタット切片媒体中で凍結する。その後、凍結切片をカットし、免疫組織化学的分析のためにスライド上に置きます。必要に応じて、組織のために使用し、100％メタノール中で脱水し、4％PFAで固定に従って全体をマウント。 </li><li>免疫組織化学的分析のために10月を除去するためにPBSに凍結切片を置く。その後、30分間、10％正常ロバ血清でセクションをブロックする。 4℃で一晩100希釈：次に1でPECAM一次抗体を追加します。翌日、10分毎にPBTで3回スライドを洗浄し、二次ロバ抗ラット594を追加し、室温で1時間インキュベートする。 注：これは、超音波およびマイクロインジェクションの最適化と調整を必要とする非常に厳しい技術です。あり、この技術に関連した非常に急な学習曲線があり、1つが堪能になる前に指導し、注射の4〜6週間が必要です。 </li></ol>

<ol>
	<li>Abrahamson, D. R., Robert, B., Hyink, D. P., St John, P. L., Daniel, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+and+formation+of+microvasculature+in+the+developing+kidney.">Origins and formation of microvasculature in the developing kidney.</a> Kidney international. Supplement. 67, S7-S11 (1998).</li><li>Sims-Lucas, S., 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=Endothelial+Progenitors+Exist+within+the+Kidney+and+Lung+Mesenchyme.">Endothelial Progenitors Exist within the Kidney and Lung Mesenchyme.</a> PloS one. 8 (6), e65993 (2013).</li><li>Dressler, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+early+kidney+specification,+development+and+patterning.">Advances in early kidney specification, development and patterning.</a> Development. 136 (23), 3863-3874 (2009).</li><li>Costantini, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+controls+and+cellular+behaviors+in+branching+morphogenesis+of+the+renal+collecting+system.">Genetic controls and cellular behaviors in branching morphogenesis of the renal collecting system.</a> Wiley Interdiscip Rev Dev Biol. 1 (5), 693-713 (2012).</li><li>Costantini, F., Kopan , R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterning+a+complex+organ:+branching+morphogenesis+and+nephron+segmentation+in+kidney+development.">Patterning a complex organ: branching morphogenesis and nephron segmentation in kidney development.</a> Dev Cell. 18 (5), 698-712 (2010).</li><li>Das, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal-epithelial+crosstalk+regulates+kidney+progenitor+cell+differentiation.">Stromal-epithelial crosstalk regulates kidney progenitor cell differentiation.</a> Nat Cell Biol. 15 (9), 1035-1044 (2013).</li><li>Rymer, C., 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=Renal+blood+flow+and+oxygenation+drive+nephron+progenitor+differentiation.">Renal blood flow and oxygenation drive nephron progenitor differentiation.</a> Am J Physiol Renal Physiol. , (2014).</li><li>Shah, S. R., 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=Embryonic+mouse+blood+flow+and+oxygen+correlate+with+early+pancreatic+differentiation.">Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation.</a> Developmental biology. 349 (2), 342-349 (2011).</li><li>Andres, A. C., 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=EphB4+receptor+tyrosine+kinase+transgenic+mice+develop+glomerulopathies+reminiscent+of+aglomerular+vascular+shunts.">EphB4 receptor tyrosine kinase transgenic mice develop glomerulopathies reminiscent of aglomerular vascular shunts.</a> Mech Dev. 120 (4), 511-516 (2003).</li><li>Wagner, R., 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=High-resolution+imaging+of+kidney+vascular+corrosion+casts+with+Nano-CT.">High-resolution imaging of kidney vascular corrosion casts with Nano-CT.</a> Microsc Microanal. 17 (2), 215-219 (2011).</li></ol>

The authors declare that they have no competing financial interests.

マイクロインジェクション麻酔と時間枠母親の麻酔に関しては、それは気流定数（2-3 L /分）と低PSIに保つことが不可欠である。鎮静剤の流れは、約1.75から2 L / minで保持されなければならない。同時に、注射が行われている時間枠を綿密に監視し、各ごみとのために制御されなければならない。各ごみための注射の手順は45分以下に抑えます。各胚が身体と関心のある器?...

胚発生中に2つの別々の、まだ同時、血管のプロセスが行われます。血管新生、血管が住宅内皮前駆細胞1,2から血管のデノボ形成であり、主要な既存の容器、および脈管形成、から成長するプロセスを。後者は、主にそれが存在しない場合に起こると考えられている間に、それぞれ、前者は、血流と同義である。 血管形成への同時、腎臓前駆細胞合成、増殖、および分化の循環的かつダイナミックなプロセスは、胎生9.5（E9.5）上で展開を開始します。この時点で、尿管芽（UB）は、周囲の後腎間葉（MM）に背側に侵入し、出産3まで続く。急速に後腎キャップ間充織を凝縮にUBの繰り返しの分岐は腎臓の機能ユニットの形成、ネフロンを開始します。 UBとnephのすべての新世代ロンは、古い世代は、それらが、その後、主に血管密度の高い環境内でさらに成熟と分化を受け、内側皮質と髄質の地域、にずれている。ドレスラーらによって証明されるように、図3に、この発生学的プロセスは、UBとMMとの間のクロストーク、および細胞外因子3-6無数のように、誘導性シグナル伝達により沈殿させる。開発膵臓と腎臓内の二つの最近調査し、細胞外の要因は、酸素緊張と血流7,8が含まれています。後者は、腎臓の発生に関連して以下でさらに詳細に説明する。 血流は、潜在的ネフロン前駆細胞の分化、並びにの他の器官形成プロセス、胚血流マッピングの精密で正確な方法で果たす役割の誘導を露出させるために不可欠である。 血流を測る別の方法は、ulの処方を含むtrasoundイメージングと樹脂が9,10を投げかける。結論的に、これらのモードは、本質的に同時に、血流との間の時間的及び空間的な並置を発表し、幹細胞の分化する能力に欠けていることが示されている。レジンキャストは、例えば、成体組織内の血管パターニングの有効なモデルを提供する、しかしそのような胚の時点と同様に未熟船、で、血管が肉眼的に未発達及び漏出性である。そのため、樹脂は、しばしば多孔質の、小さな血管内に保持することができないキャスト。 これらの見かけ上の障害のために、とりわけ、我々は超音波ガイド下腎臓発生の私たちの調査に生体内で心臓内胚トマトレクチン（TL）マイクロインジェクションに組み込むことを選んだ。この手順では、同期E11.5、E13.5、E15.5およびE17.5の時点でのマウス胚の左心室にTL溶液2.5μlので満たされたマイクロピペット取り付けられた針を案内するために超音波プローブを利用する。 E17針がより発展した胚を貫通するのに十分な強さではありませんように0.5は、最新の発達時代です。 このマイクロインジェクション法の利点は豊富である。超音波ガイド下マイクロインジェクションは、動物の鼓動する心臓への胚性左心室、ソリューションの受動的かつ制御追放、心臓や周辺組織への最小限のダメージ、との突然の心不全と死の回避内注射針の正確な位置決めが可能胚全身灌流の前に。 FITC標識TLを使用することで、すべての灌流し、血管系は、内皮頂端膜に沿ってマーカーを維持します。免疫組織化学と組み合わせて、PECAM（CD31、血小板内皮細胞接着分子）、および様々な他の血管マーカーを利用して、我々は明らかに灌流および非灌流血管を区別するだけでなく、周囲の組織の任意の異常な染色を特徴付けることができる。

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			<td>concentration 2.5&#181;L / embryo</td>
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			<td>concentration 1:200</td>
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			<td>Origio Mid Atlantic Devices</td>
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			<td style="width:164px;">03-377-20</td>
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in utero intra-cardiac tomato-lectin injections on mouse embryos to gauge renal blood flow

腎臓の血管（離れて糸球体からの）開発の形成と灌流が大幅に代役されている。脈管構造は、（主要な血管の外分岐される）は、血管形成および脈管形成（ 新生血管形成）を介して発展するにつれて、そのような生体内超音波イメージングの樹脂キャスト、および顕微解剖として灌流マッピング技術との間の密接な関係を実証するが限定されているこれら二つのプロセス及び胚内の腎臓の構造を開発。ここでは、腎臓の灌流の個体発生を測定するためにマウス胚に子宮内心臓超音波ガイド下FITC標識トマトレクチンマイクロインジェクションでの手順を説明します。トマトレクチン（TL）は、収穫胚および腎臓を通して灌流した。ネフロン前駆細胞、ネフロン構造、尿管上皮、および血管系：組織を含む様々な腎臓構造に共染色した。 E13.5から始まる大口径容器は、しかし、末梢灌流した船がunperfused残った。 E15.5およびE17.5では、小さな末梢血管だけでなく、糸球体は、灌流さになることを始めた。この実験的手法は、胚発生中の脈管構造と血流の役割を研究するために重要である。

血管形成は、腎臓の開発フローに先行 （腎臓を含む）胚組織の大部分はあっても初期胚の時点で、密な血管系（unperfusedと灌流さの両方）が含まれています。よりよいゲージに、我々は子宮内胎児の心臓内マイクロインジェクションでの方法を利用した開発腎臓内の血流を分析。 E17.5を通してE11.5で胚の心を識別するために、高解像度の超音波の使用により、および開腹?...

Watch this Scientific Journal Video about In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow at JoVE.com

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

マウス胚に子宮内 、心臓内トマトレクチン注射では腎血流量を測定する

The formation and perfusion of developing renal blood vessels (apart from glomeruli) are greatly understudied. As vasculature develops via angiogenesis ...

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Research

JoVE Journal

Developmental Biology

In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

9.6K ビュー.  Children's Hospital of Pittsburgh of UPMC. This manuscript describes a technique for visualization of the developing vasculature. Here we utilized in utero intra-cardiac FITC-labeled tomato lectin microinjections on mouse embryos. Using this technique, we delineate the perfused and unperfused vessels throughout the embryonic kidney.

ビデオ: In Utero Intra-cardiac Tomato-lectin Injections on Mouse Embryos to Gauge Renal Blood Flow

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

高周波超音波を用いて、マウス胚におけるコントラストイメージング

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

発生生物学

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

子宮外エレクトロポレーションおよびマウスの海馬組織の器官型スライス培養

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

マウス着床前胚でリネージュの仕様を研究するためのタンパク質発現の定量分析

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

loss-が及び着床前マウス胚の初期の細胞運命決定要因を調査するための機能獲得型のアプローチ

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

リコンビナーゼ媒介カセット交換を用いて、マウス胚性幹細胞における構造と機能の研究

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

ノードと走査型電子顕微鏡と蛍光抗体法マウント全体を使用して Gastrulating のマウス胚における脊索板を可視化

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by Sertoli cell junctions at the blood-testis barrier (BTB), which is the tightest tissue barrier in the mammalian body and segregates the seminiferous epithelium into two compartments, a basal and an adluminal. The BTB creates a unique microenvironment for germ cells in meiosis I/II and for the development of postmeiotic spermatids into spermatozoa via spermiogenesis. Here, we describe a reliable assay to monitor BTB integrity of mouse testis in vivo. An intact BTB blocks the diffusion of FITC-conjugated inulin from the basal to the apical compartment of the seminiferous tubules. This technique is suitable for studying gene candidates, viruses, or environmental toxicants that may affect BTB function or integrity, with an easy procedure and a minimal requirement of surgical skills compared to alternative methods.

An In Vivo Method to Study Mouse Blood-Testis Barrier Integrity

Here, we present a protocol to assess the blood-testis barrier integrity by injecting inulin-FITC into testes. This is an efficient in vivo method to study blood-testis barrier integrity that can be compromised by genetic and environmental elements.

マウス血液精巣関門の整合性を研究するIn Vivo法

Spermatogenesis is the development of spermatogonia into mature spermatozoa in the seminiferous tubules of the testis. This process is supported by ...

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 ...

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 ...

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

Postimplantation mammalian embryo culture methods have been generally inefficient and limited to brief periods after dissection out of the uterus. Platforms have been recently developed for highly robust and prolonged ex utero culture of mouse embryos from egg-cylinder stages until advanced organogenesis. These platforms enable appropriate and faithful development of pregastrulating embryos (E5.5) until the hind limb formation stage (E11). Late gastrulating embryos (E7.5) are grown in rotating bottles in these settings, while extended culture from pregastrulation stages (E5.5 or E6.5) requires a combination of static and rotating bottle cultures. In addition, sensitive regulation of O2 and CO2 concentration, gas pressure, glucose levels, and the use of a specific ex utero culture medium are critical for proper embryo development. Here, a detailed step-by-step protocol for extended ex utero mouse embryo culture is provided. The ability to grow normal mouse embryos ex utero from gastrulation to organogenesis represents a valuable tool for characterizing the effect of different experimental perturbations during embryonic development.

Ex Utero Culture of Mouse Embryos from Pregastrulation to Advanced Organogenesis

An enhanced platform for whole-embryo culture allows continuous and robust ex utero development of postimplantation mouse embryos for up to six days, from pregastrulation stages until advanced organogenesis. In this protocol, we detail the standard procedure for successful embryo culture using static plates and rotating bottle systems.

マウス胚に子宮内 、心臓内トマトレクチン注射では腎血流量を測定する

マウス胚に子宮内、心臓内トマトレクチン注射では腎血流量を測定する