我々は、神経管の画像にShlomi Krispinに感謝。特に、我々はGFPのトランスフェクション技術を開始するためのYuvalシナモンを認めます。この作品は、イスラエル科学財団、協会フランセーズcontre LESミオパシー、およびDeutche Forschungsgemeinshaft、共同研究センター488からCKへの補助金によって支えられて

1。マイクロピペットの準備我々は13.5の熱が設定された標準ナリシゲPP - 83ピペットプラーで私たちのピペットを引く。正確な設定は8〜10ミクロンのピペットの先端径を目指して、校正する必要があります。私たちは、フィラメントを含有する1.0ミリメートルと0.75〜0.78ミリメートルの内径、外径とホウケイ酸ガラス管を使用してください。ピペットは、結果的に皿の縁との接触から保護されたヒントとプラスチック製の皿に格納されています。 2。 DNAの調製目的のプラスミドを大腸菌 DH5αにトランスフェクトさ標準的な手順を用いて大腸菌 。単一のコロニーから一晩培養し、プラスミドを使用してマキシプレップキットを（任意の市販のキットが何をすべき）を精製するために使用されます。我々は、非クローン性注射用は1.0μg/μLのプラスミド濃度を使用してください。高濃度では、ピペットチップの目詰まりを増加させる。クローンのアプリケーションでは、プラスミドの濃度ははるかに低いですし（下記参照）を調整する必要があります。 pCAGG - GFPの場合は、クローンの結果を生成する最適な濃度は約0.05から0.1μg/μLのであることが判明した。プラスミドの1〜2μLは、通常、実験ごとに使用され、実験が始まる前に高速緑色の粉末（乾燥、鋭いガラスピペットの先端に付着している）は必要最小限のプラスミドに追加されます。プラスミドは、最大速度で2分間、卓上遠心機で遠心分離される。この手順では、ピペット、目詰まりの問題を軽減するようです。 3。胚の注入胚3.1準備私たちは、注射用ウズラと同様に鶏の卵を使用してください。卵は、所望の発達段階まで、その長軸上に寝そべってインキュベートする。卵は70％エタノールでswabbedと乾燥させる。卵の先端は、外科用はさみ（これが唯一の鶏の卵に必要である）と1mLの（ウズラ）または10 mLシリンジ（19G針）で卵から描かれている卵白を2mL（ひよこ）で穴を開けている。セキュリティホールは、熱いパラフィンで密封されています。 実際の注入に先立ち、ウィンドウは、外科はさみを使って卵の殻の上部に開かれます。シェルを介して粘着テープを置いて、それを切断するには、シェルから破片を減らすことができます。抗生物質を含むPBS緩衝液を卵にすぐにピペットで入れる。胚にアクセスするには、卵黄及び/または羊膜は、ニードルホルダーにマウントさ0.14ミリメートル虫ピンを使ってカットして迂回されます。非毒性インク（ペリカン＃17）アスピレーターに搭載されたキャピラリープル火を使用して、胚を視覚化するために胚盤葉の下に注入されます。 3.2マイクロピペットのロードとマウント引き手のマイクロピペットをアスピレーターにマウントされている、適切な直径の火 - プ​​ル毛細血管によってプラスミドをロードすることができます。プラスミドは必要最小限のキャピラリーを使用して、その開口部の反対側から先鋭化先端にピペットにロードされ、そして可能な限り内側から先端に近づいて描画されます。ピペットは、結果的にインジェクターがマニピュレータに搭載された手動の空気の圧力の上にロードされます。最小限の圧力は、プラスミド溶液は、ピペットの先端に到達することを可能にするために、必要に応じて、適用されます。 3.3プラスミド注入ピペットチップの目詰まりを防ぐためにいくつかの空気の圧力をかけながらピペットを卵の液体培地中に操作されます。関連する組織のドメインを刺している。先端が組織に入ると圧力の変化が頻繁に目に見える速いグリーンでプラスミド放出するのに十分です。そうでない場合は、プラスミド/高速グリーンになるまで注射器にわずかな圧力を適用して排出されます。このプロセスは、隣接する体節の例のためにまたは神経管の範囲に沿って、胚の軸に沿って繰り返すことができます。注射後、PBSを添加してもよい。ウィンドウが（パラフィルムは、特にウズラのために望ましいものではない卵、これはシェルを介して酸素交換を遮断することによって、おそらく、胚の死亡率を増加させる）粘着テープを用いて封止されている。処理された胚をインキュベーターに戻されます。換気を避け、彼らが水分と増加収量を維持するためにインキュベーターの水で小さな容器を置きます。 4。注入パラメータを調整し、成功率を監視 Asessingと様々な注入パラメータのキャリブレーションは、シングルセル解析を目指したアプリケーションにとって重要です。いないすべての注入イベントは、単一セルの成功ラベリングにつながる。高い故障率の非常に低濃度の結果ながら、高すぎるとプラスミド濃度は、非クローン性ラベリングにつながることができます。注入されたプラスミドの濃度は、クローンの注射のために最適化されている必要があります。我々は、あらかじめ決められた濃度に個々のプレップを希釈するのではなく、全体の精製されたプラスミド株式の希薄化をお勧めします。後者のおそらく、そのようなスーパーコイルプラスミドの割合として純度の変動と他のパラメータの設定によって、成功率のいくつかの差異が導入される可能性があります。 全体のマウントの観察によって成功率の評価4.1 注入された胚の初期評価は、蛍光双眼でホールマウントの観察によって行うことができます。 6〜8時間後に注射はpCAGG強化されたGFPのコンストラクトで成功率を評価するために十分である。これは、標的部位（例えば背側体節、背側神経管、外胚葉などは、図1A - Cを参照してください。）容易に明らかになるのに十分な表面的な状態で提供される。このステップでは、あまりにも多くの標識細胞あるいは、任意の蛍光細胞が欠如しているのどちらかを示す実験的なシリーズを評価するために特に有用です。さらにインキュベーションを希望する場合は、抗生物質含有PBS溶液を表示した後に卵を追加する必要がありますし、シールテープを交換する必要があります。 シリアルセクション分析によってクローンの評価4.2 成功した注射の速度を評価するための最良の手段は、注入された胚を直列に切片と標識された細胞を可視化するために免疫組織化学を使用することです。これらの条件下では、インキュベーション時間は4時間後の注射可能な限り短くすることができます。胚を固定し、パラフィン切片（またはcryosectioning）のために処理されます。ガラスにマウントされたセクションは、脱パラフィン処理し、レポーターの可視化のための免疫染色です。このようなビオチン - ストレプトアビジンのような増幅方法は、望ましいかもしれません。最後に、連続切片を注入セグメントで標識された細胞を検出するためにスキャンされます。むしろ、単一細胞よりも複数の時に最初の試験が得られる場合、十分な結果が得られるまで、プラスミド株式を希釈し、プロセスが繰り返されている必要があります。プラスミドストック溶液の2〜10倍希釈では高すぎて成功率のケースで必要になる場合があります。 それぞれのプラスミドをトランスフェクトされるため、クローンを確実に徹底したキャリブレーションを繰り返す必要があります。 5。代表的な結果胚1 2あたり6〜12のセグメントをターゲットにしながら我々は一般的に、体節ごとpCAGG - GFPの単回注射を行う。この設定では、明確なシグナルは10のうち1胚の少なくとも1つのセグメントでは、全体のマウントの観察8時間のポスト噴射により検出することができます。これらの条件下で、標識は、さらに（図1B、C）クローンであることが確認された。高DNA濃度を使用する場合、ほとんどの胚は、複数のセルをトランスフェクトされたという可能性を高め、少なくとも一つの成功注入を示すことができる。 連続切片の解析では、成功率は約10％のときクローンのトランスフェクションが達成されたことを明らかにした。これは、クローンを達成するために、技術は、定義ごとに非常に非効率的であることを意味します。一方、これらの条件下で、正のラベリングイベントの93％以上は1クローンであることがわかった。 <img src="/files/ftp_upload/2133/2133fig1.jpg" alt="図1" /> 図1。神経や骨格前駆細胞にpCAGGS - GFPのクローン注射。背側神経管のラベル（NT）（A）、dermomyotome（DM）（B）の腹ドメイン、または中央のDMシート（C）を示す（AC）横断切片では、 、単一の前駆細胞にpCAGGS - GFPのマイクロインジェクション後6hr。 （D）真皮（D）だけでなく、筋肉（M）（refを参照してください。詳細については、1）両方の誘導体を生成された中央のDM、単一の前駆細胞のクローン性のトランスフェクション後二日。

<ol>
	<li>Ben-Yair, R., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lineage+analysis+of+the+avian+dermomyotome+sheet+reveals+the+existence+of+single+cells+with+both+dermal+and+muscle+progenitor+fates.">Lineage analysis of the avian dermomyotome sheet reveals the existence of single cells with both dermal and muscle progenitor fates.</a> Development. 132, 689-701 (2005).</li><li>Ben-Yair, R., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notch+and+bone+morphogenetic+protein+differentially+act+on+dermomyotome+cells+to+generate+endothelium,+smooth,+and+striated+muscle.">Notch and bone morphogenetic protein differentially act on dermomyotome cells to generate endothelium, smooth, and striated muscle.</a> J Cell Biol. 180, 607-618 (2008).</li><li>Cinnamon, Y., Kahane, N., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+early+development+of+specific+hypaxial+muscles+from+the+ventrolateral+myotome.+.">Characterization of the early development of specific hypaxial muscles from the ventrolateral myotome. .</a> Development. 126, 4305-4315 (1999).</li><li>Stark, D. A., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+green+fluorescent+protein+as+a+single-cell+marker+in+living+embryos.">Photoactivatable green fluorescent protein as a single-cell marker in living embryos.</a> Dev Dyn. 233, 983-992 (2005).</li><li>Haas, K., Jensen, K., Sin, W. C., Foa, L., Cline, H. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+electroporation+in+Xenopus+tadpoles+in+vivo--from+single+cells+to+the+entire+brain.">Targeted electroporation in Xenopus tadpoles in vivo--from single cells to the entire brain.</a> Differentiation. 70, 148-154 (2002).</li><li>Haas, K., Sin, W. C., Javaherian, A., Li, Z., Cline, H. T. <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+electroporation+for+gene+transfer+in+vivo.">Single-cell electroporation for gene transfer in vivo.</a> Neuron. 29, 583-591 (2001).</li><li>Hewapathirane, D. S., Haas, K. <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+electroporation+in+vivo+within+the+intact+developing+brain.">Single cell electroporation in vivo within the intact developing brain.</a> J Vis Exp. , (2008).</li><li>Nagai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat Biotechnol. 20, 87-90 (2002).</li><li>Sato, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+integration+and+conditional+expression+of+electroporated+transgenes+in+chicken+embryos.">Stable integration and conditional expression of electroporated transgenes in chicken embryos.</a> Dev Biol. 305, 616-624 (2007).</li></ol>

利害の衝突は宣言されません。

上記の方法では、小細胞サブセットまたは単一の前駆細胞を3に親油性色素を提供するための前述した手法を変更したものです。それは標準的なエレクトロポレーション法に比べて3つの主な利点があります。最初に、それは（C、例えば図1Bを参照されたい）、エレクトロポレーションよりはるかに大きい空間分解能を可能にする、標的組織への信頼離散サイトで標識するための手段を...

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Pipette tubes</td>
<td>&nbsp;</td>
<td>Sutter Instrument</td>
<td>B100-75-10</td>
<td>&nbsp;</td>
<tr>
<td>Capillaries</td>
<td>&nbsp;</td>
<td>Drummond Scientific</td>
<td>2-000-100</td>
<td>&nbsp;</td>
<tr>
<td>Insect needles</td>
<td>&nbsp;</td>
<td>Watkins &amp; Doncaster</td>
<td>E6871</td>
<td>&nbsp;</td>
<tr>
<td>Pen-Strep solution</td>
<td>&nbsp;</td>
<td>Biological Industries</td>
<td>03-031-1B</td>
<td>&nbsp;</td>		</tbody>
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single cell transfection in chick embryos

発生生物学の中心テーマは、系統の多様化と分子メカニズムの解明である。これは正常な実験条件下で単一細胞の運命の徹底的な分析を伴います。この目的のために、単一の前駆細胞を標的とトランスフェクション法が前提です。私たちはトレーシング信頼性の高い系統と同様に遺伝子操作を可能にする、ここに、OVOでの鶏の組織で単一の細胞をトランスフェクトするための技術的に簡単な方法を説明します。特定の組織のドメインは、体節や神経管の中でターゲットとしている、とするDNAを単一の細胞の散発的なトランスフェクションの結果、関心の上皮に直接注入される。記者を使って、クローン集団は結果的に3日間まで遡ることができる、そして遺伝子操作クローン集団の挙動は、対照のそれと比較することができます。このメソッドは、遺伝子操作のための新たなツールと共に、ニワトリ胚のアクセシビリティを活用しています。我々は、比較とその広く使用されているエレクトロポレーション法上の利点、および可能なアプリケーションについて説明し、また提案されている追加のin vivoモデルで使用してください。我々は既存の系統のトレースと遺伝的干渉のツールのための重要な追加機能と補完するものとしてこのメ​​ソッドの使用を提唱。

Watch this Scientific Journal Video about Single Cell Transfection in Chick Embryos at JoVE.com

Single Cell Transfection in Chick Embryos

ファインチップを使用すると、我々はニワトリの体節や神経管のサブドメインにプラスミドDNAを注入するマイクロピペット。プラスミドの濃度は、単一のトランスフェクションされた細胞を生成するために調整されます。私たちは、その後、細胞はクローン集団に発展することができます。

鶏胚における単一細胞のトランスフェクション

A central theme in developmental biology is the diversification of lineages and the elucidation of underlying molecular mechanisms. This entails a ...

single-cell-transfection-in-chick-embryos

Research

JoVE Journal

Biology

11.0K ビュー.  Hadassah Medical School - Hebrew University. ファインチップを使用すると、我々はニワトリの体節や神経管のサブドメインにプラスミドDNAを注入するマイクロピペット。プラスミドの濃度は、単一のトランスフェクションされた細胞を生成するために調整されます。私たちは、その後、細胞はクローン集団に発展することができます。

ビデオ: Single Cell Transfection in Chick Embryos

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPCのマウス胚の解剖

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

生物学

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

HHステージ10ニワトリ胚のOVOエレクトロポレーションで

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

初期のニワトリ胚をex vivo（新文化）の文化のための方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

初期のニワトリ胚のin situハイブリダイゼーションダブルホールマウント

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

の電子顕微鏡のための胚の調製ショウジョウバエ胎児心臓の管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

マウスの錐体光受容体からの単一細胞サクション録音

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

ライブゼブラフィッシュ胚における二光子ベースの光活性化

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Imaging Cell Shape Change in Living Drosophila Embryos

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging. Cell shape changes in the fly are analogous to those in higher organisms, and they drive tissue morphogenesis. So, in many cases, their study has direct implications for understanding human disease (Table 1)1-5. On the sub-cellular scale, these cell shape changes are the product of activities ranging from gene expression to signal transduction, cell polarity, cytoskeletal remodeling and membrane trafficking. Thus, the Drosophila embryo provides not only the context to evaluate cell shape changes as they relate to tissue morphogenesis, but also offers a completely physiological environment to study the sub-cellular activities that shape cells.
The protocol described here is designed to image a specific cell shape change called cellularization. Cellularization is a process of dramatic plasma membrane growth, and it ultimately converts the syncytial embryo into the cellular blastoderm. That is, at interphase of mitotic cycle 14, the plasma membrane simultaneously invaginates around each of ~6000 cortically anchored nuclei to generate a sheet of primary epithelial cells. Counter to previous suggestions, cellularization is not driven by Myosin-2 contractility6, but is instead fueled largely by exocytosis of membrane from internal stores7. Thus, cellularization is an excellent system for studying membrane trafficking during cell shape changes that require plasma membrane invagination or expansion, such as cytokinesis or transverse-tubule (T-tubule) morphogenesis in muscle.
Note that this protocol is easily applied to the imaging of other cell shape changes in the fly embryo, and only requires slight adaptations such as changing the stage of embryo collection, or using "embryo glue" to mount the embryo in a specific orientation (Table 1)8-19. In all cases, the workflow is basically the same (Figure 1). Standard methods for cloning and Drosophila transgenesis are used to prepare stable fly stocks that express a protein of interest, fused to Green Fluorescent Protein (GFP) or its variants, and these flies provide a renewable source of embryos. Alternatively, fluorescent proteins/probes are directly introduced into fly embryos via straightforward micro-injection techniques9-10. Then, depending on the developmental event and cell shape change to be imaged, embryos are collected and staged by morphology on a dissecting microscope, and finally positioned and mounted for time-lapse imaging on a confocal microscope.

Imaging Cell Shape Change in Living Drosophila Embryos

Early development of the fruit fly, Drosophila melanogaster, is characterized by a number of cell shape changes that are well suited for imaging approaches. This article will describe basic tools and methods required for live confocal imaging of Drosophila embryos, and will focus on a cell shape change called cellularization.

リビングでのイメージング、細胞の形状の変更ショウジョウバエ胚

The developing Drosophila melanogaster embryo undergoes a number of cell shape changes that are highly amenable to live confocal imaging.  Cell shape ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

ゼブラフィッシュにおける単一の細胞運命のマッピング

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

顔面移植<em&gt;アフリカツメガエル</em&gt;胚

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...