著者は、デボラハリスとダニエルマッケイには特にお世話になります。この作品のサポートは、生物学科とCWRUの芸術と科学の大学で、生物科学における学部教育の支援のためのハワードヒューズの助成金番号52005866によって提供されていました。

1。イメージング手法<ol><li>それが均一に液体になるまで20〜30分55℃程度の水浴中にゼリーグリセリンを溶かす。渦巻が、気泡の形成を避けるために、ボトルを振っていない。 </li><li>ゼリーが溶けている間、きれいなガラススライド（オプション）にキムワイプを用いてグリセロールの薄層を適用する。 </li><li>薄い最初のレイヤーを作成するスライドの表面上にカットP1000ピペットチップを使用してゼリー溶けたグリセリンのピペット1から1.5 mLの。泡を避けるために慎重にピペット。 </li><li>ゼリーは、水平面上で5分間、その後5分間-20℃でスライドを配置し、胚の整列に進みます固化してみましょう。また、サランラップでスライドをカバーし、最大5日間4℃に置きます。 </li><li>ゼリーの寝具の側に70％グリセロール/ PBSまたは他の退色グリセロールベースマウントメディア（例えばSlowfadeまたはVectashield）で染色した胚の小滴をピペット。 </li><li>個別にゼリーの表面にドロップダウンリストから、それらを転送することによって胚を事前に合わせ始める。スプーンのように傾斜した面を使用して、斜めの皮下注射針を使用してそれらを転送します。 </li><li>およそ2から3列に沿って胚を置く。胚を針に陥っている場合、それを解放するために、他の胚を含むドロップダウンでそれをかき混ぜる。慎重にこの時点でそれらを合わせて時間を無駄にしないでください。列の間にスペースを残す。 </li><li>皮下注射針の助けを借りて、ゼリーの培地上に水平にスライドするだけで胚の位置を合わせます。一方、スライド、向きが同じ方向に頭と尾。列に沿って互いにそれらを平行にしてください。ツイストペアKimiwipeの切れ端で証跡として残すPBSのグリセロールの過剰を削除します。 注：過剰なグリセロールは難しいカットしてマウントすること、二つのゼリー層の分離の結果、胚がゲル内に緩く包むべきものになります。逆に、あまりにも多くのグリセロールを削除すると、乾燥や胚を平らになります。 </li><li>胚（50〜150μL、整列された数に応じて）上のゼリーの薄層を広げるためにカット黄色の先端を使用してください。 </li><li>場所の10〜20分間、-20℃の冷凍庫でスライドまたは4℃で一晩保管してください。ゼリーはカットする前に完全に剛性であることを確認します。それ以外の場合、胚のブロックは、物品税が非常に困難になります。最先端で最良の結果は、次の日に取得されます。 </li><li>解剖スコープで、整列胚の両側にゼリーを介して完全にカットするためにカミソリの刃を使用してください。ストレートカットを作るために90 °の角度でカミソリの刃を保持する。カットは、撮影の妨げになる余分なゼリーを避けるために、胚の前部と後部の極に非常に近いなされるべきである。 </li><li>カットの隣に冷たいPBT 100〜200μL（4℃、PBS +0.1％Tween20を）追加し、慎重にスパチュラを用いて胚の間にゼリーを削り取る。 注：PBTの洗剤は、胚を損傷することなく、スライドからゼリーの除去に役立ちます。 </li><li>針を使用して、さらに加工するためのクリーンなスライドに埋め込まれた胚とゼリーのブロックを転送する。 5月7日胚で小さなブロックをカット。簡単にゼリーのブロックを移動するPBTを使用してください。また、それは必要に応じて正確に、ゼリーをトリミングするための、より安定性を作成するガラス、に接続できるように乾燥したガラスの表面にブロックを設置。 </li><li>かみそりの刃と針の助けを借りてゼリーに埋め込まれた直立胚を反転します。顕微鏡下で可視化のために、使用されているスタンド顕微鏡の種類に応じて、以下の2つの手順のいずれかに進みます。 </li><li>倒立顕微鏡：場所は直立の位置に胚を、長いカバースリップの上に胚のブロックを終了する。ゼリーの最低額と胚の側は客観的との緊密な接触になることを確認してください。 </li><li>正立顕微鏡：&quot;サポートする&quot;として使用されると、&quot;サポートする&quot;との間にグリセロールの胚のブロックを配置するために瞬間接着剤で接着4本の＃1カバーグラスで2つのスタックを作る。長いカバースリップを使用してスタックを埋める。 </li><li>共焦点分析のために、あなたが画像のすべての方法を胚を介して、または部分的にしか処理できるかどうかを調べるために使用される目的の作動距離を確認してください。たとえば、D.キイロ胚の長さは〜0.55 mmを持っていると完全にツァイス20倍プランアポまたは40倍LDネオフルーアの対物レンズを用いて画像化することができます。次のWebサイトは、利用可能なツァイスの目標に関する情報を持って、あなたの作動距離に応じて目標を検索するオプションを与える：https://www.micro-shop.zeiss.com/us/us_en/objektive.phpを</li></ol> 2。代表的な結果胚はそのまま残っているので、この方法は、胚のサイズと遺伝子発現パターン間の距離の正確な測定を行うために使用することができます。図1では、我々は核 ​​染色DAPI（青）、抗背タンパク質（赤）、SOGのmRNA（黄色）とSNAのmRNA（緑）で染色した胚盤葉期胚を示しています。形態学的プロセッSES、中胚葉の陥入（図2A、B）と中胚葉細胞（図3と4）の移行などもこの方法を使用して視覚化することができます。 DVの軸に沿って異なるZ -位置は、図2-4に示すように、焦点面を変更することにより得ることができます。 <img src="/files/ftp_upload/2175/2175fig1.jpg" alt="図1" /> 図1。胚盤葉の段階で無傷の胚の直立光学スライス。mRNAとタンパク質のための二重染色。 situプローブの（SOGとSNA）と一次抗体（抗背側）で染色したタンパク質の標的mRNAは、図に示されている。 DAPIは核を標識するために使用されていました。背の発現は核でありながら、SOGとSNAの発現は、細胞質内にapically配置されていることに注意してください。核に位置してSOGの新生転写物からの強い信号はまた、この倍率で見ることができます。 <img src="/files/ftp_upload/2175/2175fig2.jpg" alt="図2" /> 図2。無傷gastrulating胚の直立像。mRNAのプローブは、使用されており、それぞれの色が図に示されている。 A）、（SNA、緑のSOGとDPP、）、、この段階では同じフッ素で染色された3つの遺伝子を注意して空間的に分離されたドメインで表されます。 invaginating中胚葉は、SNAを表現する、神経芽細胞層はINDを表現し、SOGの表現はまだ神経系の腹側の領域に存在している間、外胚葉は、DPPを表現しています。 B）同じ胚の深い焦点面では、我々は強くSNAを表現invaginating中胚葉の基底に注意してください、しかし、その先端部は、低いレベルを表している。 <img src="/files/ftp_upload/2175/2175fig3.jpg" alt="図3" /> 図3。 。 カタツムリ、INDとDPPのmRNA（緑）、背側蛋白質（赤）、 胚芽のバンドとそのまま胚の直立像は、SOG RNA（黄色）を拡張 。核はDAPI（青）で染色した。胚の頭部に近いA）フォーカルプレーン。同じ胚のトランクの地域でのB）もう一つの焦点面。横方向の移行が（図4を比較）の胚帯の拡張胚の両側に腹側正中線に近い嘘前に中胚葉細胞の質量に注意してください。剥離神経芽細胞は、緑色（INDおよびSNA）のラベルが付いています。 <img src="/files/ftp_upload/2175/2175fig4.jpg" alt="図4" /> 図4。胚帯の拡張時に無傷の胚の直立像 SOG RNA（黄色）、。 カタツムリ、INDとDPPのmRNA（緑）、背側蛋白質（赤）。 A）上皮（矢印）から剥離神経芽細胞（注）、（緑）この段階ではINDとSNAの両方を染色した。 SOGの制限された発現は腹側正中（黄色）にも注意してください。この段階でDPPの発現は、胚の側面（アスタリスク）で見ることができます。 B）同じ胚の光学セクションでは、半ば後方部に相当する胚の長さ、の終わり近くに示します。腹側正中の細胞は、SOGのために染色されています。

<ol>
	<li>Ay, A., Fakhouri, W. D., Chiu, C., Arnosti, D. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image+processing+and+analysis+for+quantifying+gene+expression+from+early+Drosophila+embryos.">Image processing and analysis for quantifying gene expression from early Drosophila embryos.</a> Tissue Engineering. 14, 1517-1526 (2008).</li><li>Gr&#946;hans, J., Wieschaus, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+link+between+morphogenesis+and+cell+division+during+formation+of+the+ventral+furrow+in+Drosophila.">A genetic link between morphogenesis and cell division during formation of the ventral furrow in Drosophila.</a> Cell. 101, 523-531 (2000).</li><li>Kosman, D., Mizutani, C. M., Lemons, D., Cox, W. G., McGinnis, W., Bier, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+detection+of+RNA+expression+in+Drosophila+embryos.">Multiplex detection of RNA expression in Drosophila embryos.</a> Science. 305, 846-846 (2004).</li><li>Witzberger, M. M., Fitzpatrick, J. A. J., Crowley, J. C., Minden, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=End-on+imaging:+a+new+perspective+on+dorsoventral+development+in+Drosophila+embryos.">End-on imaging: a new perspective on dorsoventral development in Drosophila embryos.</a> Developmental Dynamics. 237, 3252-3259 (2008).</li><li>Zander, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On+mounting+delicate+bryophytes+in+glycerol.">On mounting delicate bryophytes in glycerol.</a> The Bryologist. 100, 380-382 (1997).</li><li>Liberman, L. M., Reeves, G. T., Stathopoulos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+imaging+of+the+Dorsal+nuclear+gradient+reveals+limitations+to+threshold-dependent+patterning+in+Drosophila.">Quantitative imaging of the Dorsal nuclear gradient reveals limitations to threshold-dependent patterning in Drosophila.</a> Proc Natl Acad Sci U S A. 106 (52), 22317-2222 (2009).</li></ol>

利害の衝突は宣言されません。

それは、スライス2または通常蛍光stainingsに対して有害であるミクロトーム処理、ために埋め込 ​​まれたメディアの使用のいずれかの慎重な手の郭清を必要とするので、 ショウジョウバエの胚の断面の画像を取ることは、挑戦することができます。また、共焦点顕微鏡で作られたZ -スタックは、断面の画像の3D再構成を作るために使用することができます。しかし、正確な三次元再構成と光退色問題のあるなるためにいくつかの薄い共焦点スライスを作るために時間がかかるがそれです。縦にマウントされているイメージング胚もタンパク質またはmRNA発現レベル1の定量化を目的と蛍光シグナルの正確な測定を行う複雑胚の深いセクションに到達したときに発生する光の散乱であるもう一つの懸念。たとえ2光子共焦点顕微鏡で、光散乱はまだ限られるため、サンプルの最適な透明性のためにマウントメディアの選択を行った胚の真ん中に卵黄物質の不透明度、に起因する問題です。 これらの問題を回避するために、垂直に位置決め胚のイメージング&quot;に関するエンド&quot;と呼ばれる新しい技術は、最近、画像の両方のライブと固定試料4に開発されました。本稿では、 その場プローブ3 で複数で汚れている任意のサイズと形状の固定された胚や組織の簡単な準備と可視化を可能にする縦置きでマウント胚のための新しいプロトコルを開発しました。我々の手法は、その中に整列胚を包むために使用されるゼラチン状の一貫性と取り付けメディアを使用して構成されています。埋め込まれた胚を含むこのマウントメディアのカットブロックは共焦点顕微鏡、任意のタイプのイメージングの前に直立に配置することができます。 ゼリーに胚を埋め込むと垂直に配置することによって、我々は、胚の背腹軸に沿って細胞が同時に記載されている共焦点顕微鏡を使用して、水平方向の断面を活用することができます。これは、光散乱および縦置き胚の従来のZ -スタック再建で発生する蛍光染料の光退色の問題以来、定量化の目的のために大幅な改善は、現在解消されています。反対側の背腹の位置にあるセルが同時に同じ焦点Z位置で結像されているのでこのように、背腹軸に沿った遺伝子発現やタンパク質レベルの定量は、正確です。さらに、記載された方法は、私たちはDVの軸6に沿って異なる位置にあるセルを視覚化するために、複雑な計算アンロールの方法を使用せずに3 - D胚から背腹軸を介して完全な2次元画像を取得することができます。 重要なステップのいくつかは、ゼリーの2つの層の間の分裂を避けるために、胚を合わせ後に余分なグリセリンを除去含まれています。しかし、サンプルがあまりにも脱水の場合は、結果として得られる画像は不格好になると不正な形態を持つことになります。胚の周りゼリーの培地は角度ではなく、ストレートカットされている場合に加えて、、、結果として得られるイメージは、90度以外の角度で胚の不正確なポジショニングのために、形状の小さい丸とより多くの卵子の表示されることがあります。ゼリーは前方/後方端部に胚に密接に十分にカットされていない場合は最後に、、それは客観の作動距離は主にゼリーではなく胚に集中するために使用されるので、適切な画像を得ることは不可能である。 イメージング後半gastrulating胚は慎重にそれらを整列させる前に、スコープの下に関心の段階をソートするときに必要になることがありますもう一つの重要なステップ。通常、胚は縦位置で最も簡単に認識できる形態学的特性に応じてステージングされます。 この方法で作ることができる別の変更の中に蛍光染料の光退色を防ぐためにゼリーの二層にアンチフェード試薬（例えば、p -フェニレンジアミンまたはPPD）に含めることです。

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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>Glycerin Jelly mounting media</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences Company</td>
<td>17998-10</td>
<td>Contains low concentration of phenol as preservative and should be used in hood or in well ventilated area. Recipe for making your own media is described in Zander, 1997.</td>
</tr>
<tr>
<td>Zeiss LSM 700 Confocal</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>&nbsp;</td>
<td>The following objectives were used: Plan-Apo 20x 0.8 M27 (WD 0.55mm); EC Plan-Neofluar 40x 1.3 oil (WD 0.21mm); LD Plan-Neofluar 0.6 Korr 40X (WD 2.9mm).</td>
</tr>
<tr>
<td>Phosphate buffered saline with 0.1% Tween 20 (PBT)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Glass microscope slides</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>12-544-7</td>
<td>&nbsp;</td>
<tr>
<td>Glass cover slips</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>72204-02</td>
<td>Size 1 &frac12; (specific for the objectives used in this work).</td>
</tr>
<tr>
<td>Glass cover slips (for making supports)</td>
<td>&nbsp;</td>
<td>Fisherfinest</td>
<td>12-544-10</td>
<td>Use four coverslips glued with superglue. </td>
</tr>
<tr>
<td>Dissection microscope</td>
<td>&nbsp;</td>
<td>M5 Wild Heerbrugg Wild Makroskop</td>
<td>M420</td>
<td>&nbsp;</td>
<tr>
<td>Razor blade</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Steel back single edge industrial blades</td>
</tr>
<tr>
<td>Spatula</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>21-401-10</td>
<td>&nbsp;</td>
<tr>
<td>Hypodermic needles</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>1482610</td>
<td>26 Gauge</td>
</tr>
<tr>
<td>Pipettes</td>
<td>&nbsp;</td>
<td>Labnet</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

upright imaging of drosophila embryos

いくつかのよく知られた形態形成の勾配と細胞の動きが背/腹軸に沿って起こる<em&gt;ショウジョウバエ</em&gt;胚。しかし、そのようなプロセスを表示するために使用される現在の技術は幾分限られている。以下のプロトコールは、固定とラベルの付いたマウントするための新しい手法を説明します<em&gt;ショウジョウバエ</em&gt;共焦点イメージングと冠状表示するための胚。このメソッドは二つのグリセリンゼリーマウントメディアの層、そして直立に配置イメージングゼリーストリップの間に胚を埋め込むから成ります。胚を挟むための最初のステップは、スライド上のグリセリンゼリーの薄い寝具をすることです。次に、胚は、慎重にこの表面上に整列し、ゼリーの二層で覆われている。第二層が固化した後、ゼリーのストリップを切断し、イメージングのために直立反転されています。選択肢は、使用する顕微鏡のスタンドのタイプに応じて胚を視覚化するために記載されています。背腹軸に沿ってすべてのセルが単一の共焦点Z -平面内に結像されているので、私たちの手法は、正確な測定と光退色せずに蛍光シグナルまたは縦置き胚の3D再構成に共通の光散乱の比較が可能になります。

Watch this Scientific Journal Video about Upright Imaging of Drosophila Embryos at JoVE.com

Upright Imaging of Drosophila Embryos

ここでは、ステンドグラスの取り付けプロトコルを提示<em&gt;ショウジョウバエ</em&gt;共焦点顕微鏡を用いて断面のイメージングを可能にする直立姿勢における胚。

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 ...

Nanyang Technological University

Research

JoVE Journal

Biology

Upright Imaging of Drosophila Embryos

9.3K ビュー.  Case Western Reserve University. ここでは、ステンドグラスの取り付けプロトコルを提示ショウジョウバエ共焦点顕微鏡を用いて断面のイメージングを可能にする直立姿勢における胚。

ビデオ: Upright Imaging 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 ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

収穫と準備ショウジョウバエ</em電気生理学的記録とその他の手続のための>胚

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

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 ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

のライブイメージングキイロショウジョウバエ胚性血球移行

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

In-vivo Centrifugation of Drosophila Embryos

A major strategy for purifying and isolating different types of intracellular organelles is to separate them from each other based on differences in buoyant density. However, when cells are disrupted prior to centrifugation, proteins and organelles in this non-native environment often inappropriately stick to each other. Here we describe a method to separate organelles by density in intact, living Drosophila embryos. Early embryos before cellularization are harvested from population cages, and their outer egg shells are removed by treatment with 50% bleach. Embryos are then transferred to a small agar plate and inserted, posterior end first, into small vertical holes in the agar. The plates containing embedded embryos are centrifuged for 30 min at 3000g. The agar supports the embryos and keeps them in a defined orientation. Afterwards, the embryos are dug out of the agar with a blunt needle. Centrifugation separates major organelles into distinct layers, a stratification easily visible by bright-field microscopy. A number of fluorescent markers are available to confirm successful stratification in living embryos. Proteins associated with certain organelles will be enriched in a particular layer, demonstrating colocalization. Individual layers can be recovered for biochemical analysis or transplantation into donor eggs. This technique is applicable for organelle separation in other large cells, including the eggs and oocytes of diverse species.

In-vivo Centrifugation of Drosophila Embryos

We describe a method to separate organelles by density in living Drosophila embryos. Embryos are embedded in agar and centrifuged. This technique yields reproducible separation of major organelles along the anterior-posterior embryo axis. This method facilitates colocalization experiments and yields organelle fractions for biochemical analysis and transplantation experiments.

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 ...

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 ...

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 ...