我々はそれぞれ、Sciara coprophilaとIlyanassa obsoletaに生体内の遠心分離をテストするために材料を供給するためのスーザンGerbi、ハイジスミス、およびデビッドランバートに感謝。私たちは、テクニックや原稿上のコメントのWelte氏研究室のメンバーに感謝。 生体内の遠心分離アッセイの開発と改良はMAWにNIGMS助成金GM64687によってサポートされていました。図に示されている画像。図1、図。 5E、Fは、以前は次の2を出版され、その許諾を得てここに転載されています。

<ol>
	<li>Wieschaus, E., N&#252;sslein-Volhard, C., Roberts, D. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Looking+at+embryos.">Looking at embryos.</a> Drosophila: A Practical Approach. , 179-214 (1998).</li><li>Cermelli, S., Guo, Y., Gross, S. P., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lipid+droplet+proteome+reveals+that+droplets+are+a+protein+storage+depot.">The lipid droplet proteome reveals that droplets are a protein storage depot.</a> Curr Biol. 16, 1783-1795 (2006).</li><li>Kiehart, D. P., Crawford, J. M., Montague, R. A., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+microinjection+of+Drosophila+embryos.">Quantitative microinjection of Drosophila embryos.</a> Drosophila Protocols. , 345-359 (2000).</li><li>LaJeunesse, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+new+Drosophila+markers+of+intracellular+membranes.">Three new Drosophila markers of intracellular membranes.</a> Biotechniques. 36 (784-788), 790-790 (2004).</li><li>Schuh, M., Lehner, C. F., Heidmann, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+Drosophila+CID/CENP-A+and+CENP-C+into+centromeres+during+early+embryonic+anaphase.">Incorporation of Drosophila CID/CENP-A and CENP-C into centromeres during early embryonic anaphase.</a> Curr Biol. 17, 237-243 (2007).</li><li>Clarkson, M., Saint, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+His2AvDGFP+fusion+gene+complements+a+lethal+His2AvD+mutant+allele+and+provides+an+in+vivo+marker+for+Drosophila+chromosome+behavior.">A His2AvDGFP fusion gene complements a lethal His2AvD mutant allele and provides an in vivo marker for Drosophila chromosome behavior.</a> DNA Cell Biol. 18, 457-462 (1999).</li><li>Welte, M. A., Gross, S. P., Postner, M., Block, S. M., Wieschaus, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+regulation+of+vesicle+transport+in+Drosophila+embryos:+forces+and+kinetics.">Developmental regulation of vesicle transport in Drosophila embryos: forces and kinetics.</a> Cell. 92, 547-557 (1998).</li><li>Tran, S. L., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=unpublished+observations.">unpublished observations.</a> , .</li><li>Rothwell, W. F., Sullivan, W., Sullivan, W., Ashburner, M., Hawley, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+analysis+of+Drosophila+embryos.">Fluorescent analysis of Drosophila embryos.</a> Drosophila Protocols. , 141-157 (2000).</li><li>Guo, Y., Jangi, S., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organelle-specific+Control+of+Intracellular+Transport:+Distinctly+Targeted+Isoforms+of+the+Regulator+Klar.">Organelle-specific Control of Intracellular Transport: Distinctly Targeted Isoforms of the Regulator Klar.</a> Mol Biol Cell. 16, 1406-1416 (2005).</li><li>Meyer, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overlapping+Functions+of+Argonaute+Proteins+in+Patterning+and+Morphogenesis+of+Drosophila+Embryos.">Overlapping Functions of Argonaute Proteins in Patterning and Morphogenesis of Drosophila Embryos.</a> PLoS Genet. 2, 1224-1239 (2006).</li><li>Shubeita, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+motor+copy+number+on+the+intracellular+transport+of+kinesin-1-driven+lipid+droplets.">Consequences of motor copy number on the intracellular transport of kinesin-1-driven lipid droplets.</a> Cell. 135, 1098-1107 (2008).</li><li>Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+lipid-droplet+transport+by+the+Perilipin+homologue+LSD2.">Regulation of lipid-droplet transport by the Perilipin homologue LSD2.</a> Curr. Biol. 15, 1266-1275 (2005).</li><li>Phadnis, N., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpublished+Observations.">Unpublished Observations.</a> , .</li><li>Phadnis, N., Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpublished+Observations.">Unpublished Observations.</a> , .</li><li>Morgan, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+XXII:+The+redistribution+of+the+visible+materials+of+the+egg+by+centrifuging.">Chapter XXII: The redistribution of the visible materials of the egg by centrifuging.</a> Experimental Embryology. , (1927).</li><li>Welte, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unpublished+Observations.">Unpublished Observations.</a> , .</li><li>Bownes, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormal+oogenesis+and+embryogenesis+resulting+from+centrifuging+Drosophila+melanogaster+females.">Abnormal oogenesis and embryogenesis resulting from centrifuging Drosophila melanogaster females.</a> J Embryol Exp Morphol. 40, 65-81 (1977).</li></ol>

利害の衝突は宣言されません。

重要なステップ 寒天の濃度が十分な大きさであることを確認してください。それが低すぎる場合は、寒天は、遠心分離中に崩壊し、胚が破裂したり、ランダムに配向されます。寒天の濃度は（ステップ4.3の過剰TSSの不十分な乾燥または不十分な除去）をわずかに低すぎるまたは寒天が濡れている場合であれば、胚は、遠心分離中にそれらの穴からフロートと不適切指向になります。寒天があまりにも薄く注がれている場合、それはまた、遠心分離中に崩壊したり、割れの原因となる。 オルガネラが個々の細胞の数千の中で隔離さになるので、 胚の細胞化の段階を超えて進んでいないことを確認してください。そうしないと、細胞内小器官の分離では（図5C）は動作しません。一つの細胞の段階では若い胚では、細胞内小器官は、大きな距離を移動する自由であり、その特徴的な密度に応じて蓄積する。古すぎる胚を分析避けるために、（i）を、女性は古い卵を（ステップ2.4​​）レイアウトするために事前にコレクションを含む（ⅱ）（ⅲ）、（ステップ2.5）3時間以内のために胚を回収することを確認してください視覚的に寒天に埋め込むため細胞化ステージの前に胚を選択する（ステップ4.2）、（IV）細胞化（ステップ4.6）の最後まで開発を防ぐために、短い時間を埋め込む維持、および（v）（ステップ5.6）正しく層しなかった胚を破棄。 遠心分離の時間を出し惜しみしないでください。胚の一部も遠心分離のわずか10分後に素敵なレイヤーが表示されますが、分離は胚から胚に矛盾している。 30分のスピンは非常に一貫した結果を与える。遠心分離が終了した後も、長く遠心時間は、長く層が安定しています。これは、使用前にさらに胚を処理するのに時間がかかりますで、これらのアプリケーションにとって重要です（例えば、彼らは移植実験のために処理または共焦点分析のためにマウントする必要がある場合）。 実行可​​能な変更 卵の収集と絨毛膜の除去 、多くの異なるプロトコルのステップ2と3 1、3、9のために存在し、動作するだけでなく、ものがここで使用される胚の若者である限り説明し、卵の収率が高くなる、との割合ミス段階的な胚は最小限に抑えられます。 後端は寒天と前端に埋もれていることをすべての胚はそのような前後位置合わせのプロトコルに配向は（図3）までです。このオリエンテーションでは、それが最後の遠心分離（図1、5）中に指されるランドマークとして卵門を使用することが可能となるように、一貫性のために良いです。しかし、実際に、それは、明視野顕微鏡による脂質滴と卵黄の層の異なる外観で遠心分離胚の方向を選ぶのは簡単です。その後、どちらかの端で胚をマウントすることが可能になり、この柔軟性は、実装工程をスピードアップします。 未延伸遠心分離プロトコルの中で最も時間がかかり、技術的に困難な側面は、寒天の胚（ステップ4）を取り付けています。それは、（一回穴に挿入するには、一度遠心分離した後、それを回復する）二回各胚に触れる必要があります。別の方法として、一つはTSSで満たされたマイクロチューブにステップ4.1に胚を転送することができます。これらのチューブを遠心（13,000 rpmで、10〜30分間）で遠心分離されている場合、胚意志もよく、通常、層10-13この変化は1つが非常に迅速に、同時に胚の数百を処理することができます。しかし、階層化は、一貫していません。胚の割合は、上記のプロトコルに適用されるよりもはるかに高い遠心力（〜16,000 G）にもかかわらず、異なる層を開発していません。より挑戦的な胚の向きが可変であり、そして一つの主要な胚軸に様々な角度で分離を観察できるという事実です。このばらつきは、実験者は、特定胚を遠心管に配置されたか、遠心分離後にソートする必要があります。それは確実にそれを行うには多くの練習とより多くの判断を必要とするものの胚の中で最も密度の高い部分のためのマーカーとして、卵黄の自家蛍光を使用すると、これは、多くの場合可能です。サンプルで十分な胚がある場合は、一つは胚の図のように配置するとき、何が起こっているにそれらのインスタンスをソートすることができるかもしれません。 1。 その卵の殻の胚の遠心分離は、それは最初の2絨毛膜を除去せずに、手順4の寒天に胚を埋め込 ​​むことが可能です。このアプローチでは、コレクションプレート上胚はハロカーボンのオイルの代わりにステップ3.2（ハロカーボンは、絨毛膜の半透明に変化）で50％の漂白剤で覆われている。適切な段階の胚を選択し、ピンセットを使用して、新しい寒天プレートに転送されます。ピンセットでのみ卵のフィラメントをつかんで、損傷を胚の適切なが回避される。胚は前卵のフィラメントは、寒天の穴の外まで付着して、から4.8ステップ4.4のように埋め込まれている。胚を遠心分離し、ステップ5.4のように寒天に掘らされた後に卵の殻は、50％の漂白剤の処理により除去される。卵の殻の存在下で遠心分離する（ステップ6.2）固定後devitellinizationの速度を改善するかもしれませんが、遠心分離のための適切な段階での選択（ステップ4.2）より困難です。 アプリケーション胚の遠心分離は、特定のタンパク質が特定の主要な細胞小器官に局在するかどうかをテストするために、すなわち、局在の実験のために特に有用である。例えば、クラータンパク質が初期胚脂質滴上に存在する、まだこの局在は、無傷の胚で実証するために困難である。クラー涙点と脂肪滴の両方が除外するのは難しいスプリアス共局在を作り、胚の周辺部10に豊富に存在するので、一部では、これは。しかしながら、遠心分離は、異なる層に脂肪滴を集中し、従って、クラー信号と共局在は10明らかになる。 同様の分析は、明確に特定のヒストンのような驚くべき例を含む脂肪滴8、13-15、2とタンパク質が遍在存在する場合には他のタンパク質の局在を示したが脂肪滴8に富んでいる。胚が発達段階（ステップ4.2）で視覚的に選択できるように、それは脂肪滴8、15に蛋白質の分化段階募集を検出することも可能です。脂肪滴は、視覚的に明確な&quot;キャップ&quot;に蓄積するので、脂肪滴と共局在するためのテストは特に簡単ですが、それ以上のマーカーまたは染色がこの層を識別するために必要ありません。実際には、理由その使いやすさと潜在的に印象的なビジュアルの結果（図1、図5、D、E）から、私たちの研究室では、候補タンパク質が脂肪滴に局在しているかどうか最初のテストとして、現在日常的にこの手法を採用しています。原則として、同様の共局在の研究は、図に示すように、他の小器官のために働く必要があります。その分布遠心分離胚の文書化が完了していない他の人のための可能性1、（細胞内寄生体を含む）。 遠心分離は、タイトなバンドに小器官を絞っているので、それはこれらの細胞小器官に富む画分の分離を容易にします。このアプローチはすでにドナー胚2に移植のために脂肪滴を回復するために使用されています。また、生化学的に濃縮画分（例えば、固定15日以降の胚からの層を切断することによって）分析することが可能であるかもしれません。 方法は、遠心分離は多くの異なる種の卵（カエル、線虫、ホヤ、環形動物、ウニ、貝類、そして刺胞動物を含む）16を層別化するために使用できるように、 ショウジョウバエの胚を超えてアプリケーションを持ってここで説明する。我々自身の研究室では、イエバエイエバエ 2の胚については、上記のプロトコルを使用しているとキノコバエSciara coprophila 17とカタツムリIlyanassa obsoleta 17日から胚の解析で未延伸遠心分離を用いている。最後に、この方法は、胚に限定されるものではありません。遠心はまた、卵母細胞（例えば、 ショウジョウバエ 2、18、トウワタバグOncopeltusアカハタ 17）などの巨大な細胞を、層別化すると便利です。異なる試料の場合は、正確な遠心分離の条件は、最適化する必要があります：例えば、3000gで10分、効率的にイエバエ 2の胚を層別化するのに十分である、とはるかに長い間遠心分離した場合、胚は、固定および免疫染色時にバラバラにする傾向がある。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>60x15 mm Petri dishes</td>
<td>&nbsp;</td>
<td>Becton Dickinson</td>
<td>#35-1007</td>
<td>From Falcon</td>
</tr>
<tr>
<td>Wire mesh</td>
<td>&nbsp;</td>
<td>Small Parts</td>
<td>CX-0150</td>
<td>stainless steel type 304; mesh size #150 x150, wire diameter: 0.0026 inches</td>
</tr>
<tr>
<td>Tungsten needles</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>10130-10</td>
<td>0.25 mm</td>
</tr>
<tr>
<td>Moria Nickel Plated Pin/Needle Holder</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26016-12</td>
<td>&nbsp;</td>
<tr>
<td>Fly Cages</td>
<td>&nbsp;</td>
<td>Genesee Scientific</td>
<td>59-100 or 59-101</td>
<td>For 65 mm or 100 mm diameter Petri dishes, respectively</td>
</tr>
<tr>
<td>Halocarbon Oil 27</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H8773</td>
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in-vivo centrifugation of drosophila embryos

細胞内小器官の様々なタイプを精製し、単離するための主要な戦略は、浮遊密度の差に基づいて、お互いからそれらを分離することです。しかし、細胞を前に遠心分離に中断されている場合、この非ネイティブ環境でのタンパク質や細胞小器官は、しばしば不適切にお互いに固執する。ここではそのまま、生きてショウジョウバエの胚で密度によって細胞小器官を分離する方法を説明します。細胞化前の初期胚は、人口のケージから収穫され、その外側の卵の殻は、50％の漂白剤の処理により除去されています。胚はその後、寒天に小さな縦の穴に、最初に小さな寒天プレートと挿入、後端に転送されます。埋め込まれた胚を含むプレートは3000グラムで30分間遠心分離されています。寒天は、胚をサポートしており、定義された方向でそれらを保持します。その後、胚を鈍針で寒天に掘られている。 遠心分離は、明視野顕微鏡による階層化が容易に目に見える、別個の層に主要な細胞内小器官を分離する。蛍光マーカーの数は、生きた胚で成功した成層を確認するために用意されています。特定のオルガネラに関連するタンパク質が共局在を示す、特定の層に濃縮されます。個々の層は、提供された卵子に生化学的解析や移植のために回収することができます。この技法は、多様な種の卵や卵母細胞を含む他の大規模な細胞、内オルガネラの分離に適用可能です。

Watch this Scientific Journal Video about In-vivo Centrifugation of Drosophila Embryos at JoVE.com

In-vivo Centrifugation of Drosophila Embryos

我々は生体内での密度によって細胞小器官を分離する方法を説明します<em&gt;ショウジョウバエ</em&gt;胚。胚を寒天に埋め込まれ、遠心分離されています。この手法は、前後の胚軸に沿って主要な細胞小器官の再現可能な分離が得られます。このメソッドは、生化学的解析と移植の実験のために共局在の実験と収量オルガネラの分画を容易にします。

ショウジョウバエの胚の生体内の遠心

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in ...

in-vivo-centrifugation-of-drosophila-embryos

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JoVE Journal

Biology

In-vivo Centrifugation of Drosophila Embryos

18.3K ビュー.  University of Rochester. 我々は生体内での密度によって細胞小器官を分離する方法を説明しますショウジョウバエ胚。胚を寒天に埋め込まれ、遠心分離されています。この手法は、前後の胚軸に沿って主要な細胞小器官の再現可能な分離が得られます。このメソッドは、生化学的解析と移植の実験のために共局在の実験と収量オルガネラの分画を容易にします。

ビデオ: In-vivo Centrifugation of Drosophila Embryos

Preparation of Neuronal Cultures from Midgastrula Stage Drosophila Embryos

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos and their dechorionation using bleach are demonstrated. Using a glass pipet attached to a mouth suction tube, we illustrate the removal of all cells from single embryos. The method for dispersing cells from each embyro into a small (5 l) drop of medium on an uncoated glass coverslip is demonstrated. A view through the microscope at 1 hour after plating illustrates the preferred cell density. Most of the cells that survive when grown in defined medium are neuroblasts that divide one or more times in culture before extending neuritic processes by 12-24 hours. A view through the microscope illustrates the level of neurite outgrowth and branching expected in a healthy culture at 2 days in vitro. The cultures are grown in a simple bicarbonate based defined medium, in a 5% CO2 incubator at 22-24&deg;C. Neuritic processes continue to elaborate over the first week in culture and when they make contact with neurites from neighboring cells they often form functional synaptic connections. Neurons in these cultures express voltage-gated sodium, calcium, and potassium channels and are electrically excitable. This culture system is useful for studying molecular genetic and environmental factors that regulate neuronal differentiation, excitability, and synapse formation/function.

This video demonstrates the preparation of primary neuronal cultures from midgastrula stage Drosophila embryos. Views of live cultures show cells 1 hour after plating and differentiated neurons after 2 days of growth in a bicarbonate-based defined medium. The neurons are electrically excitable and form synaptic connections.

Midgastrulaステージショウジョウバエの胚からの神経培養の準備

This video illustrates the procedure for making primary neuronal cultures from midgastrula stage Drosophila embryos. The methods for collecting embryos ...

生物学

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

の調製ショウジョウバエ</emハンギングドロッププロトコルを使用してライブイメージングのための>胚

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Visualizing developing organ formation as well as progession and treatment of disease often heavily relies on the ability to optically interrogate molecular and functional changes in intact living organisms. Most existing optical imaging methods are inadequate for imaging at dimensions that lie between the penetration limits of modern optical microscopy (0.5-1mm) and the diffusion-imposed limits of optical macroscopy (&gt;1cm) [1]. Thus, many important model organisms, e.g. insects, animal embryos or small animal extremities, remain inaccessible for in-vivo optical imaging. 
Although there is increasing interest towards the development of nanometer-resolution optical imaging methods, there have not been many successful efforts in improving the imaging penetration depth. The ability to perform in-vivo imaging beyond microscopy limits is in fact met with the difficulties associated with photon scattering present in tissues. Recent efforts to image entire embryos for example [2,3] require special chemical treatment of the specimen, to clear them from scattering, a procedure that makes them suitable only for post-mortem imaging. These methods however evidence the need for imaging larger specimens than the ones usually allowed by two-photon or confocal microscopy, especially in developmental biology and in drug discovery.
We have developed a new optical imaging technique named Mesoscopic Fluorescence Tomography [4], which appropriate for non-invasive in-vivo imaging at dimensions of 1mm-5mm. The method exchanges resolution for penetration depth, but offers unprecedented tomographic imaging performance and it has been developed to add time as a new dimension in developmental biology observations (and possibly other areas of biological research) by imparting the ability to image the evolution of fluorescence-tagged responses over time. As such it can accelerate studies of morphological or functional dependencies on gene mutations or external stimuli, and can importantly, capture the complete picture of development or tissue function by allowing longitudinal time-lapse visualization of the same, developing organism.
The technique utilizes a modified laboratory microscope and multi-projection illumination to collect data at 360-degree projections. It applies the Fermi simplification to Fokker-Plank solution of the photon transport equation, combined with geometrical optics principles in order to build a realistic inversion scheme suitable for mesoscopic range. This allows in-vivo whole-body visualization of non-transparent three-dimensional structures in samples up to several millimeters in size.
We have demonstrated the in-vivo performance of the technique by imaging three-dimensional structures of developing Drosophila tissues in-vivo and by following the morphogenesis of the wings in the opaque Drosophila pupae in real time over six consecutive hours.

Mesoscopic Fluorescence Tomography for In-vivo Imaging of Developing Drosophila

Mesoscopic fluorescence tomography operates beyond the penetration limits of tissue-sectioning fluorescence microscopy. The technique is based on multi-projection illumination and a photon transport description. We demonstrate in-vivo whole-body 3D visualization of the morphogenesis of GFP-expressing wing imaginal discs in Drosophila melanogaster.

のためのメゾスコピック蛍光トモグラフィー生体内</em開発の>イメージングショウジョウバエ</em

Visualizing  developing organ formation as well as progession and treatment of disease often  heavily relies on the ability to optically interrogate ...

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 ...

Upright Imaging of Drosophila Embryos

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current techniques used to view such processes are somewhat limited. The following protocol describes a new technique for mounting fixed and labeled Drosophila embryos for coronal viewing with confocal imaging. This method consists of embedding embryos between two layers of glycerin jelly mounting media, and imaging jelly strips positioned upright. The first step for sandwiching the embryos is to make a thin bedding of glycerin jelly on a slide. Next, embryos are carefully aligned on this surface and covered with a second layer of jelly. After the second layer is solidified, strips of jelly are cut and flipped upright for imaging. Alternatives are described for visualizing the embryos depending upon the type of microscope stand to be used. Since all cells along the dorsal-ventral axis are imaged within a single confocal Z-plane, our method allows precise measurement and comparison of fluorescent signals without photobleaching or light scattering common to 3D reconstructions of longitudinally mounted embryos.

Upright Imaging of Drosophila Embryos

Here we present a mounting protocol for stained Drosophila embryos in an upright position that allows imaging of cross-sections using Confocal microscopy.

の直立イメージングショウジョウバエ胚

Several well-known morphogenetic gradients and cellular movements occur along the dorsal/ventral axis of the Drosophila embryo. However, the current ...

Primary Cell Cultures from Drosophila Gastrula Embryos

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos. In brief, a large amount of staged embryos from young and healthy flies are collected, sterilized, and then physically dissociated into a single cell suspension using a glass homogenizer. After being plated on culture plates or chamber slides at an appropriate density in culture medium, these cells can further differentiate into several morphologically-distinct cell types, which can be identified by their specific cell markers. Furthermore, we present conditions for treating these cells with double stranded (ds) RNAs to elicit gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. The ability to carry out effective RNAi perturbation, together with other molecular, biochemical, cell imaging analyses, will allow a variety of questions to be answered in Drosophila primary cells, especially those related to differentiated muscle and neuronal cells.

Primary Cell Cultures from Drosophila Gastrula Embryos

We provide a detailed protocol for preparing primary cells dissociated from Drosophila embryos. The ability to carry out the effective RNAi perturbation, together with other molecular, biochemical and cell imaging methods will allow a variety of questions to be addressed in Drosophila primary cells.

から初代細胞培養ショウジョウバエ原腸胚

Here we describe a method for preparing and culturing primary cells dissociated from Drosophila gastrula embryos.   In brief, a large amount of staged ...

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal lines have enabled biological processes to be studied in the context of a living, and in some instances even behaving, organism. In this protocol we will describe how to anesthetize intact Drosophila larvae, using the volatile anesthetic desflurane, to follow the development and plasticity of synaptic populations at sub-cellular resolution1-3. While other useful methods to anesthetize Drosophila melanogaster larvae have been previously described4,5,6,7,8, the protocol presented herein demonstrates significant improvements due to the following combined key features: (1) A very high degree of anesthetization; even the heart beat is arrested allowing for lateral resolution of up to 150 nm1, (2) a high survival rate of &gt; 90% per anesthetization cycle, permitting the recording of more than five time-points over a period of hours to days2 and (3) a high sensitivity enabling us in 2 instances to study the dynamics of proteins expressed at physiological levels. In detail, we were able to visualize the postsynaptic glutamate receptor subunit GluR-IIA expressed via the endogenous promoter1 in stable transgenic lines and the exon trap line FasII-GFP1. (4) In contrast to other methods4,7 the larvae can be imaged not only alive, but also intact (i.e. non-dissected) allowing observation to occur over a number of days1. The accompanying video details the function of individual parts of the in vivo imaging chamber2,3, the correct mounting of the larvae, the anesthetization procedure, how to re-identify specific positions within a larva and the safe removal of the larvae from the imaging chamber.

In vivo Imaging of Intact Drosophila Larvae at Sub-cellular Resolution

This protocol describes a reliable method for anesthetization and imaging of intact Drosophila melanogaster larvae. We have utilized the volatile anesthetic desflurane to allow for repetitive imaging at sub-cellular resolution and re-identification of structures for up to a few days1.

サブセルラー解像度で手つかずのショウジョウバエの幼虫の in vivoイメージングで

Recent improvements in optical imaging, genetically encoded fluorophores and genetic tools allowing efficient establishment of desired transgenic animal ...

RNAi Interference by dsRNA Injection into Drosophila Embryos

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements and tools for genetic manipulation have become available in Drosophila 2. Soon after the initial discovery by Frie and Mello 3 that double stranded RNA can be used to knockdown the activity of individual genes in Caenorhabditis elegans, RNA interference (RNAi) was shown to provide a powerful reverse genetic approach to analyze gene functions in Drosophila organ development 4, 5.
Many organs, including lung, kidney, liver, and vascular system, are composed of branched tubular networks that transport vital fluids or gases 6, 7. The analysis of Drosophila tracheal formation provides an excellent model system to study the morphogenesis of other tubular organs 8. The Berkeley Drosophila genome project has revealed hundreds of genes that are expressed in the tracheal system. To study the molecular and cellular mechanism of tube formation, the challenge is to understand the roles of these genes in tracheal development. Here, we described a detailed method of dsRNA injection into Drosophila embryo to knockdown individual gene expression. We successfully knocked down endogenous dysfusion(dys) gene expression by dsRNA injection. Dys is a bHLH-PAS protein expressed in tracheal fusion cells, and it is required for tracheal branch fusion 9, 10. dys-RNAi completely eliminated dys expression and resulted in tracheal fusion defect. This relatively simple method provides a tool to identify genes requried for tissure and organ development in Drosophila.

RNAi Interference by dsRNA Injection into Drosophila Embryos

RNA interference has been proven very effective to analyze gene function in Drosophila tracheal development. A detailed protocol used by Jiang lab to inject dsRNA into fly embryos to knockdown gene expression is illustrated. This technique has the potential for screening genes required for tissue and organ development in Drosophila.

dsRNAをインジェクションによるRNAiの干渉にショウジョウバエ胚

Genetic screening is one of the most powerful methods available for gaining insights into complex biological process 1. Over the years many improvements ...

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 ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

キネシンの単離及び精製からショウジョウバエ胚

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...