Name: a rapid and efficient method to dissect pupal wings of drosophila suitable for immunodetections or pcr assays
Uploaded: 2017-12-30T14:00:00.000+00:00
Duration: 7 min 11 s
Description: Watch this Scientific Journal Video about A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays at JoVE.com

CSIC マリアナ ・ ディ ・ Doménico の共焦点顕微鏡と彼女のテクニカル サポートをこのプロジェクトに与えられた財政援助のために感謝したいと思いますホセ レオナルドあった彼の原稿の修正; のためにルチアーノ ・ コレア、ビデオの作成に彼の献身のために貸出ビデオのための彼女の声と本研究で使用される株式を提供するブルーミントンショウジョウバエストック センターのナタリア Rosano。イザベル ゲレロによってモノクローナル抗 ptc Apa1 は、発育の後援の下から発達研究ハイブリドーマ銀行 (DSHB) が取得・保有アイオワの大学、生物科学専攻、アイオワ ・ シティ、IA 52242。

日 1
1. 収集と養殖の蛹。
<ol>
	<li>培養瓶からウェット絵筆を使用して前蛹や蛹殻形成過程 (APF) 後 0 h を削除し、開発期間をほぼ完全に新しい瓶にそれらを置く (2.1 および 2.2 を参照)。 
	注:養殖の標準プロトコルを使用して、ハエの健全な人口を維持しなければなりません。卵がレイアウトは、3 つまたは 4 日後、大人は個人の最適な開発を許可するバイアルから削除できます。3 齢幼虫が蛹化を開始するまで、彼らは一定の温度に保たれます。幼虫/蛹の転移は 0 h APF を考慮イラガ前蛹の形成によってマークされます。イラガ前蛹は、前気門を突出の円形、長方形には不動の白い幼虫です。</li>
</ol>
日 2
2. 転送、郭清と固定。
<ol>
	<li>転送 (2 h 開発時間が前に完了) 蛹、絵筆に両面テープを顕微鏡スライドを遵守。背側面と頭部を持つ行が同じ方向を向いて終了フォームに蛹を配置します。</li>
	<li>顕微鏡スライドを一定の温度に戻り、蛹 (すなわち24 30 h APF) 適切な発達時期を完了するを許可します。 
	注:両面テープで顕微鏡スライドを蛹の場合 (下記参照) を削除する一時的な郭清のプラットフォームとなります。(バイアル) 外顕微鏡スライドの時間の経過は、鉗子で簡単に壊れやすい、蛹のケースを乾燥するのに役立ちます。</li>
	<li>鉗子を使用して蛹のケースから、蓋を取り外します。</li>
	<li>(ビデオで示すように)、鉗子で箱を壊す尾末に蛹の側面に沿って行を作るします。適切な照明と蛹の翼のヒンジのすぐ上の領域を明らかになり蛹の内部形状を検出することが可能です。このスペースは、蛹を傷つけることがなく、開口部を実行するためのガイドとして機能します。</li>
	<li>開いた枠でそれらを拾い、両面テープがそれらを保持する反対側にそれらを運ぶケースの部分を削除します。解剖の終わりに蛹はほとんど「裸」、ケースの小さい部分だけをカバーする後部のヒントになります。 注: 上部の尾側端の場合の少量を残しての目的は、両面テープでスライドにケースからスムーズなリリースを確保するためです。このアクションの背後にある理由ですのであまりにも多くを削除するかどうかはケースの蛹を損傷大幅リスクします。その一方であまりを削除する場合蛹簡単に来ないケースの無料。</li>
	<li>場合の残りの部分 (鉗子) と押し下げます。この動きは、頭と胸部に従うと後続の手順で転送する優先位置にとどまること、蛹の前端を発生します。</li>
	<li>両面テープで新しいスライドを取るし、蛹や蛹の行にそれを反転します。蛹の破損を防ぐために顕微鏡の助けを借りて運動を観察することによってこのアプローチを実行することをお勧めします。</li>
	<li>蛹のテープに固執し、場合の残りの部分から、蛹を取除くためにスライドがゆるやかに少し押します。顕微鏡のスライドを反転: 蛹は自分の頭のレベルで背側に立ち往生されます/胸部領域。</li>
	<li>4% パラホルムアルデヒドですべての裸蛹をカバーし、10 分を待ちます。我々 の経験で定着剤と時間のこの経過はしっかり、少ない粘着性と扱いやすいキューティクルの一貫性を変更するのに役立ちます。 
	注:リボ核酸 (RNA) の抽出を実行するには、RNase で作られたリン酸緩衝生理食塩水 (PBS) の代替パラホルムアルデヒドは無料の水、各肢のヒンジ ポイント近く (はさみ借りて) カットを作り蛹の翼を取り外します。鉗子を使用して、ためチオシアン酸とフェノール試薬 1 mL の微量遠心チューブに蛹翅を転送します。50 80 羽の合計を収集した後に、RNA 抽出10,11を実行するまで-80 ° C で遠心チューブを格納します。</li>
	<li>またはその近くに翼のヒンジ領域で小切開を行います。翼上皮に到達する定着剤を許可します。ピペット (p200) フラッシュ定着性を使用して、翼の上。フラッシュを交換するタイミング、汚染された新たにこだわる。フラッシュ後、に 5 分間漬けます翼を維持します。 
	注:この演習は汚染組織の大半を離れてオフに役立ちますが、いくつかの血液細胞と脂肪滴は翼の背側と腹側の上皮層の間に閉じ込められて残ることができる (図 1A参照)。</li>
	<li>(ビデオで示すように)、キューティクル国境から鉗子で涙穴を拡大し、表皮嚢から翼上皮のリリースを有効にします。翼の組織をリリースすると、一度は、固定の 30 分を完了するパラホルムアルデヒド溶液中残します。</li>
	<li>1 pbst; 満ちている 4 流したプラスチックの皿に固定翼を転送 (トリトン X-100 0.05%)。4 ° C で一晩ストア</li>
</ol>
日 3
3. 一次抗体の孵化。
<ol>
	<li>PBST で蛹の翼の培養組織を permeabilize (トリトン X-100 0.2%) 穏やかな液体中の動きを容易にする揺動のプラットフォームを使用して 15 分。 
	注:サンプルをマウントするまでロッキング プラットフォームを使用して手順を実行する必要があります。</li>
	<li>PBSTA を使用してサンプルをブロック (BSA 3%、トリトン X-100 0.1%) 1 時間。</li>
	<li>同じバッファーを使用して一次抗体 (マウス抗-Ptc は、1: 100) を希釈し、4 ° C で一晩蛹の翼を孵化させなさい</li>
</ol>
日 4
4. 二次抗体の孵化。
<ol>
	<li>洗浄サンプル 4 × 10 の分を各 PBST (トリトン X-100 0.05%)</li>
	<li>(例えば、抗マウス螢光色素に共役) 二次抗体とインキュベート PBSTA で適切な濃度に希釈して (BSA 3%、トリトン X-100 0.1%) 室温 2 時間、暗闇の中で。 
	注:それが適切な場合、組織を counterstain に 4', 6-diamidino-2-phenylindole (DAPI) やファロイジンを使用します。これらのプローブを信号の重なりを防ぐために蛍光色素の発光波長を考慮して二次抗体に追加します。</li>
	<li>洗浄サンプル 4 × 10 の分を各 PBST (トリトン X-100 0.05%)</li>
</ol>
5. 取付
<ol>
	<li>彼らは正方形の頂点であるかのように、顕微鏡スライドの透明なマニキュアの 4 ドットを配置することによってサンプル スライドを準備します。</li></ol>彼らがここ、coverslips 休むことは、ポイントになる組織を粉砕避けるために助けます。
	<li>メディア (80% グリセロール/トリス-HCl) 角 (ドット) のいずれかに触れることがなく爪のポーランド語で作られたドットの正方形の中心でのマウントの場所 30 μ L。黄色のトリミングされたチップとピペットを取る、いくつかのマウントのメディアをロードし、いくつか蛹翼で描きます。</li>
	<li>蛹の翼を顕微鏡スライドにメディアをマウントのドットに転送します。ヒントの助けを借りて取り付けメディアの普及し、フローティングからそれらを防ぐために蛹の翼をシンクします。組織の形態は、カバーガラスに配置する場合、翼が横ばいの場合に保持されます。カバーガラスを配置する前に、空気の泡を削除します。</li>
	<li>気泡の発生を防ぐためにしようとしているサンプルに coverslip を下げます。この動きは、鉗子の助けを借りて行うことができます。</li>

<ol>
	<li>Serrano, N., O'Farrell, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limb+morphogenesis:+connections+between+patterning+and+growth.">Limb morphogenesis: connections between patterning and growth.</a> Curr Biol. 7 (3), R186-R195 (1997).</li><li>Blair, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartments+and+appendage+development+in+Drosophila.">Compartments and appendage development in Drosophila.</a> Bioessays. 17 (4), 299-309 (1995).</li><li>Klein, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wing+disc+development+in+the+fly:+the+early+stages.">Wing disc development in the fly: the early stages.</a> Curr Opin Genet Dev. 11 (4), 470-475 (2001).</li><li>Beira, J. V., Paro, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+legacy+of+Drosophila+imaginal+discs.">The legacy of Drosophila imaginal discs.</a> Chromosoma. 125 (4), 573-592 (2016).</li><li>De Celis, J. F., Diaz-Benjumea, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+basis+for+vein+pattern+variations+in+insect+wings.">Developmental basis for vein pattern variations in insect wings.</a> Int J Dev Biol. 47 (7-8), 653-663 (2003).</li><li>Blair, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wing+vein+patterning+in+Drosophila+and+the+analysis+of+intercellular+signaling.">Wing vein patterning in Drosophila and the analysis of intercellular signaling.</a> Annu Rev Cell Dev Biol. 23, 293-319 (2007).</li><li>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=Drawing+lines+in+the+Drosophila+wing:+initiation+of+wing+vein+development.">Drawing lines in the Drosophila wing: initiation of wing vein development.</a> Curr Opin Genet Dev. 10 (4), 393-398 (2000).</li><li>De Celis, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+the+Drosophila+wing:+The+development+of+the+veins.">Pattern formation in the Drosophila wing: The development of the veins.</a> Bioessays. 25 (5), 443-451 (2003).</li><li>Classen, A. K., Aigouy, B., Giangrande, A., Eaton, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+Drosophila+pupal+wing+morphogenesis.">Imaging Drosophila pupal wing morphogenesis.</a> Methods Mol Biol. 420, 265-275 (2008).</li><li>Bolatto, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+and+temporal+distribution+of+Patched-related+protein+in+the+Drosophila+embryo.">Spatial and temporal distribution of Patched-related protein in the Drosophila embryo.</a> Gene Expr Patterns. 19 (1-2), 120-128 (2015).</li><li>Zuniga, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+encoding+novel+secreted+and+transmembrane+proteins+are+temporally+and+spatially+regulated+during+Drosophila+melanogaster+embryogenesis.">Genes encoding novel secreted and transmembrane proteins are temporally and spatially regulated during Drosophila melanogaster embryogenesis.</a> BMC Biol. 7, 61 (2009).</li></ol>

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郭清はこのビデオで説明されているメソッドを使用して、技術の多くの種類の別の開発段階からショウジョウバエ蛹翼の高品質なサンプルを準備できます (例えば、蛍光抗体法、PCR の試金、cDNA 合成in vitro における翼の開発)。このメソッドの面で関与する科学者は 24-30 h APF を使用しています。しかし若いまたは古い蛹の郭清に不可欠なステップ以内の障害はないはず。変...

ショウジョウバエ羽翅成虫原基から開発されています。これらの翼のディスクの原初は、胚上皮から鞘に収めた 20 30 成虫細胞の小さなグループとして萌芽期の開発の間に脇に置きます。有糸分裂がディスク1,2,3、その数 (約 50000 細胞) を上げると翼を形成特性の特定の時間に成虫細胞に発生する幼虫が 3 令期に高まる一方 4。細胞の増殖、彼らの自己折りたたみコンパクトなスパイラルの結果、尿細管上皮をファッションします。蛹化、ディスク上皮は 2 層フォーム翼に望遠鏡を縦方向の静脈は、翼発達5,6の中に 2 回発生するそれらの間の腔として起動。
常に静脈の配列が同一のパターン;静脈形成内すべての変化を簡単に識別できる変異非常に目に見える (例えば行方不明または余分な静脈、静脈の位置等の変更。) になります。興味深いことに、表現型変化、通常知られているまたは新しいコンポーネント翼開発に関連するシグナルの突然変異の証拠です。位置の決定、静脈と intervein 地域を維持する役割を果たしている経路の 1 つはハリネズミ (Hh) 経路6,7,8です。
モデル システムとして蛹翅の重要性を考えると、それはで動作するように良い品質のサンプルを得ることが重要なります。過去には、ショウジョウバエのハネを解剖するためのプロトコルを公開している科学者実行可能なサンプルを達成するために適切なガイドを与えていません。研究者の指導なし 1 つ明確にプロセスを視覚化することはできません。これらの代替解剖アプローチの蛹は周囲の組織から分離すること、2 つの翼に分割、瓦礫の撤去には繰り返し洗浄します。この種類の自由の翼を残す方法クレームの汚染しますが、以前の経験のために、処理が遅くなります、壊れやすい翼9の構造を損なうことのより高いチャンスがあります。
ここで説明する手順は、蛹翅サンプルの特定の蛋白質や謄本バイオアセッテイ プロトコルまたはポリメラーゼの連鎖反応 (PCR) の試金を使用しての検出に適してを取得する高速かつ効率的な方法を見つけるための需要によって開発されました。式およびパッチ適用 (Ptc または Hh 経路の正規受容体) のローカリゼーション、これを説明するためにが正常に完全なを実行する関連する詳細情報を含むプロトコルを提供する蛹翅のサンプルで検出されました。プロシージャ。

<table><tbody><tr><td>Stereomicroscope with cool light illumination</td><td>Nikon&amp;nbsp;</td><td>SMZ1000</td><td></td></tr><tr><td>Rocking platform</td><td>Biometra (WT 16)</td><td>042-500</td><td></td></tr><tr><td>Microscope slides</td><td>StarFrost</td><td>8037/1</td><td></td></tr><tr><td>Cover glasses (24 x 32 mm)</td><td>Deltalab</td><td>D102432</td><td></td></tr><tr><td>Forceps</td><td>F.S.T</td><td>11252-00</td><td></td></tr><tr><td>Scissors</td><td>Lawton</td><td>63-1400</td><td></td></tr><tr><td>Multi-well plastic dish</td><td>Thermo Scientific</td><td>144444</td><td></td></tr><tr><td>p100 or p200 pipette</td><td>Finnpipette (Labsystems)</td><td>9400130</td><td></td></tr><tr><td>1X PBS (Dulbecco)</td><td>Gibco</td><td>21600-010</td><td></td></tr><tr><td>4% paraformaldehyde&amp;nbsp;</td><td>Sigma</td><td>158127</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>BSA</td><td>Bio Basic INC.</td><td>9048-46-8</td><td></td></tr><tr><td>Glycerol</td><td>Mallinckrodt</td><td>5092</td><td></td></tr><tr><td>Tris</td><td>Amresco</td><td>497</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Ambion</td><td>15596026</td><td></td></tr><tr><td>DEPC treated water</td><td>MO.BIO</td><td>17011-200</td><td></td></tr><tr><td>Alexa Fluor 546 goat anti-mouse IgG (H+L), 2 mg/mL&lt;br/&gt; (working dilution 1/800)</td><td>Invitrogen</td><td>A11030</td><td></td></tr><tr><td>Monoclonal anti-Ptc (Apa-1) 54 &amp;mu;g/mL&lt;br/&gt; (working dilution 1/100)</td><td>Developmental Studies Hybridoma Bank (DSHB)</td><td></td><td>By Isabel Gerrero from DSHB under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, IA 52242.&amp;nbsp;</td></tr><tr><td>DAPI, 1 mg/mL&lt;br/&gt; (working dilution 1/5000)</td><td>Invitrogen</td><td>62248</td><td></td></tr><tr><td>Phalloidin conjugated to Alexa 488, 300 U/1.5 mL methanol&lt;br/&gt; (1/600 working dilution)</td><td>Invitrogen</td><td>A12379</td><td></td></tr><tr><td>Canton S (fly stock)</td><td>Bloomington Drosophila Stock Center&amp;nbsp;</td><td></td><td></td></tr></tbody></table>

a rapid and efficient method to dissect pupal wings of drosophila suitable for immunodetections or pcr assays

キイロショウジョウバエの翼の開発は、組織レベルでの形態形成を研究するための理想的なモデルです。これらの肢は、翅成虫原基萌芽期の開発の間に形成の名前セルのグループから開発します。幼虫の段階で成虫原基の成長、細胞の数の増加と単層上皮構造を形成します。蛹のケース内成虫ディスクはうち芽、翼の今後のマージンになりますラインに沿って二重に折る。このプロセス中には、縦 primodia 静脈静脈翼の将来の背側と腹側の表面に細胞を起源としています。蛹の段階で各表面の静脈細胞のストライプのタイトなチューブを生成するために通信します。同時にクロス静脈の形成を開始します。
適切な分子マーカーの助けを借りて、その開発中に翼を構成する主要な要素を識別することが可能です。このため、正確にこの構造体の成績証明書または蛋白質を検出する能力は、彼らの豊かさと翼の開発プロセスに関連するローカリゼーションを勉強するため重要です。
手順は、蛹の段階で翼を分析する方法の詳細な手順を提供して、蛹の翼を操作することに焦点を当ててを紹介します。蛹の組織の郭清は 3 齢幼虫での対応よりも難しくなります。これは、迅速かつ効率的な高品質なサンプルを取得するこの方法を開発した理由です。Immunostain とマウントする方法の詳細について蛋白質または細胞のコンポーネントの可視化できるように、これらの翼サンプルは、プロトコルで提供されます。少しの専門知識を持つ 8-10 高品質蛹翼を短時間で収集することが可能です。

プロトコルでは、翼のおなじみの形態を保持する蛹翅試料の採取の簡単な方法を提供しています。これらの製剤から 2-d または 3-D、配布または翼の分化タンパク質の濃縮を調査として投影できる画像のスタックを取得することが可能です。同様のプロトコル (2.9 の注を参照) は、相補的デオキシリボ核酸 (cDNA) の PCR の反作用で使用されるテンプレートを合成に必要な?...

Watch this Scientific Journal Video about A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays at JoVE.com

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

正確にショウジョウバエのティッシュの成績証明書または蛋白質を検出する能力は、その豊かさとローカライズ開発プロセスに関連の勉強にとって重要です。ここ蛹翼を分析する簡単な手順の説明です。これらの翼は、いくつかのテクニック(免疫組織化学、PCR 法等)のサンプルとして使用できます。

Immunodetections または PCR の試金に適したショウジョウバエの蛹の翼を解剖する迅速かつ効率的な方法

Wing development in Drosophila melanogaster is an ideal model for studying morphogenesis at tissue level. These appendages develop from a group of cells ...

a-rapid-efficient-method-to-dissect-pupal-wings-drosophila-suitable

Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

8.0K ビュー.  Laboratorio de Biología del Desarrollo. 正確にショウジョウバエのティッシュの成績証明書または蛋白質を検出する能力は、その豊かさとローカライズ開発プロセスに関連の勉強にとって重要です。ここ蛹翼を分析する簡単な手順の説明です。これらの翼は、いくつかのテクニック(免疫組織化学、PCR 法等)のサンプルとして使用できます。

ビデオ: A Rapid and Efficient Method to Dissect Pupal Wings of Drosophila Suitable for Immunodetections or PCR Assays

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and mechanobiology, which all influence muscle development and myofibrillogenesis. Omics data, such as those generated by mass spectrometry or deep sequencing, can provide important mechanistic insights into these biological processes. For such approaches, it is beneficial to analyze tissue-specific samples to increase both selectivity and specificity of the omics fingerprints. Here we present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched muscle samples for omics applications. We first describe how to dissect flight muscles at early pupal stages (&#60;48 h after puparium formation [APF]), when the muscles are discernable by green fluorescent protein (GFP) labeling. We then describe how to dissect muscles from late pupae (&#62;48 h APF) or adults, when muscles are distinguishable under a dissecting microscope. The accompanying video protocol will make these technically demanding dissections more widely accessible to the muscle and Drosophila research communities. For RNA applications, we assay the quantity and quality of RNA that can be isolated at different time points and with different approaches. We further show that Bruno1 (Bru1) is necessary for a temporal shift in myosin heavy chain (Mhc) splicing, demonstrating that dissected muscles can be used for mRNA-Seq, mass spectrometry, and reverse transcription polymerase chain reaction (RT-PCR) applications. This dissection protocol will help promote tissue-specific omics analyses and can be generally applied to study multiple biological aspects of myogenesis.

Dissection of Drosophila melanogaster Flight Muscles for Omics Approaches

Drosophila flight muscle is a powerful model to study transcriptional regulation, alternative splicing, metabolism, and mechanobiology. We present a protocol for dissection of fluorescent-labeled flight muscle from live pupae to generate highly enriched samples ideal for proteomics and deep-sequencing. These samples can offer important mechanistic insights into diverse aspects of muscle development.

オミクスアプローチに対するショウジョウバエメラノガスター飛行筋肉の解剖

Drosophila flight muscle is a powerful model to study diverse processes such as transcriptional regulation, alternative splicing, metabolism, and ...

遺伝学

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

でProprioceptorsの可視化<em&gt;ショウジョウバエ</em&gt;幼虫および蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

神経科学

Dissection and Staining of Drosophila Pupal Ovaries

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a pupal case. The challenge of dissecting pupal ovaries also lies in their physical location within the pupa: the ovaries are surrounded by fat body cells inside the pupal abdomen, and these fat cells must be removed to allow for proper antibody staining. To overcome these challenges, this protocol utilizes customized Pasteur pipets to extract fat body cells from the pupal abdomen. Moreover, a chambered coverglass is used in place of a microcentrifuge tube during the staining process to improve visibility of the pupae. However, despite these and other advantages of the tools used in this protocol, successful execution of these techniques may still involve several days of practice due to the small size of pupal ovaries. The techniques outlined in this protocol could be applied to time course experiments in which ovaries are analyzed at various stages of pupal development.

Dissection and Staining of Drosophila Pupal Ovaries

The Drosophila ovary is an excellent model system for studying stem cell niche development. Though methods for dissecting larval and adult ovaries have been published, pupal ovary dissections require different techniques that have not been published in detail. Here we outline a protocol for dissecting, staining, and mounting pupal ovaries.

郭清およびショウジョウバエ蛹卵巣の汚損

Unlike adult Drosophila ovaries, pupal ovaries are relatively difficult to access and examine due to their small size, translucence, and encasing within a ...

発生生物学

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and behavioral evolution. Drosophila olfactory tissues house the olfactory receptor neurons (ORNs) that detect volatile chemical cues in addition to hydro- and thermo-sensory neurons. In this protocol, we describe the dissection of developing peripheral olfactory tissue of the adult Drosophila species. We first describe how to stage and age Drosophila larvae, followed by the dissection of the antennal disc from early pupal stages, followed by the dissection of the antennae from mid-pupal stages and adults. We also show methods where preparations can be utilized in molecular techniques, such as the RNA extraction for qRT-PCR, RNAseq, or immunohistochemistry. These methods can also be applied to other Drosophila species after species-specific pupal development times are determined, and respective stages are calculated for appropriate aging.

Preparing Developing Peripheral Olfactory Tissue for Molecular and Immunohistochemical Analysis in Drosophila

Here, we present a protocol to stage and dissect developing olfactory tissue from Drosophila species. The dissected tissue can later be used for molecular analyses, such as quantitative RT-PCR (Reverse Transcription-Polymerase Chain Reaction) or RNA sequencing (RNAseq), as well as in vivo analyses such as immunohistochemistry or in situ hybridization.

周辺嗅覚組織の開発を準備する分子とショウジョウバエの免疫組織化学的解析

The olfactory system of Drosophila is a widely used system in developmental neurobiology, systems neuroscience, as well as neurophysiology, behavior, and ...

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a reliable protocol for the dissection of the delicate Drosophila pupal retina. Our surgical approach utilizes readily-available microdissection tools to open pupae and precisely extract eye-brain complexes. These can be fixed, subjected to immunohistochemistry, and retinas then mounted onto microscope slides and imaged if the goal is to detect cellular or subcellular structures. Alternatively, unfixed retinas can be isolated from brain tissue, lysed in appropriate buffers and utilized for protein gel electrophoresis or mRNA extraction (to assess protein or gene expression, respectively). Significant practice and patience may be required to master the microdissection protocol described, but once mastered, the protocol enables relatively quick isolation of mainly undamaged retinas.

Dissection of the Drosophila Pupal Retina for Immunohistochemistry, Western Analysis, and RNA Isolation

This paper presents a surgical method for dissecting Drosophila pupal retinas along with protocols for the processing of tissue for immunohistochemistry, western analysis, and RNA-extraction.&#160;

免疫組織化学、西部の分析、および RNA の隔離のためのショウジョウバエ蛹網膜の解剖

The Drosophila pupal retina provides an excellent model system for the study of morphogenetic processes during development. In this paper, we present a ...

Imaging Dpp Release from a Drosophila Wing Disc

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in multicellular organisms. The TGF-&#946; superfamily includes TGF-&#946;, bone morphogenetic protein (BMPs), Activins, Growth and Differentiation Factors, and Nodals. It has long been known that the amount of ligand exposed to cells is important for its effects. It was thought that long-range concentration gradients set up embryonic pattern. However, recently it has become clear that the timing of exposure to these ligands is also important for their downstream transcriptional consequences. A TGF-&#946; superfamily ligand cannot have a developmental consequence until it is released from the cell in which it was produced. Until recently, it was difficult to determine when these ligands were released from cells. Here we show how to measure the release of a Drosophila BMP called Decapentaplegic (Dpp) from the cells of the wing primordium or wing disc. This method could be modified for other systems or signaling ligands.

Imaging Dpp Release from a Drosophila Wing Disc

The timing of exposure to ligands may impact their developmental consequences. Here we show how to image release of a Drosophila bone morphogenetic protein (BMP) called Dpp from cells of the wing disc.

ショウジョウバレウィングディスクからのイメージングDppリリース

The transforming Growth Factor-beta (TGF-&#946;) superfamily is essential for early embryonic patterning and development of adult structures in ...

Drosophila Pupal Abdomen Immunohistochemistry

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic morphologies 1-3. During pupation, which spans approximately 96 hours (at 25 &deg;C), proliferating populations of imaginal cells replace the larval epidermis to generate the adult abdominal segments. These imaginal cells, born during embryogenesis, exist as lateral pairs of histoblast nests in each abdominal segment of the larvae. Four pairs of histoblast nests give rise to the adult dorsal cuticle (anterior and posterior dorsal nests), the ventral cuticle (ventral nests) and the spiracles associated with each segment (spiracle nests) 4. Upon puparation, these diploid cells (distinguishable by size from the larger polyploid larval epidermal cells- LECs) begin a stereotypical process of proliferation, migration and replacement of the LECs. Various molecular and genetic tools can be employed to investigate the contributions of genetic pathways involved in morphogenesis of the adult abdomen. Ultimate adult phenotypes are typically analyzed following dissection of adult abdominal cuticles. However, investigation of the underlying molecular processes requires immunohistochemical analyses of the pupal epithelium, which present unique challenges. Temporally dynamic morphogenesis and the interactions of two distinct epithelial populations (larval and imaginal) generate a fragile tissue prone to excessive cell loss during dissection and subsequent processing. We have developed methods of dissection, fixation, mounting and imaging of the Drosophila pupal abdominem epithelium for immunohistochemical studies that generate consistent high quality samples suitable for confocal or standard fluorescent microscopy.

Drosophila Pupal Abdomen Immunohistochemistry

Antibody staining of the Drosophila pupae can enhance genetic analyses of adult abdominal developmental genetics. We present our protocol for dissection, fixation and antibody staining of staged Drosophila pupal abdomen.

ショウジョウバエの蛹の腹部の免疫組織化学

The Drosophila pupal abdomen is an established model system for the study of epithelial morphogenesis and the development of sexually dimorphic ...

生物学

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, innate immune cells perform a number of essential functions, such as fighting infection, clearing necrotic debris, and stimulating matrix deposition. In order to fully understand the diverse signaling events that regulate this immune response, it is crucial to observe the complex behaviors of (and interactions that occur between) multiple cell lineages in vivo, and in real-time, with the high spatio-temporal resolution. The optical translucency and the genetic tractability of Drosophila embryos have established Drosophila as an invaluable model to live-image and dissect fundamental aspects of inflammatory cell behavior, including mechanisms of developmental dispersal, clearance of apoptotic corpses&#160;and/or microbial pathogens, and recruitment to wounds. However, more recent work has now demonstrated that employing a much later stage in the Drosophila lifecycle &#8211; the Drosophila pupa &#8211; offers a number of distinct advantages, including improved RNAi efficiency, longer imaging periods, and significantly greater immune cell numbers. Here we describe a protocol for imaging wound repair and the associated inflammatory response at the high spatio-temporal resolution in live Drosophila pupae. To follow the dynamics of both re-epithelialization and inflammation, we use a number of specific in vivo fluorescent markers for both the epithelium and innate immune cells. We also demonstrate the effectiveness of photo-convertible fluorophores, such as Kaede, for following the specific immune cell subsets, to track their behavior as they migrate to, and resolve from, the injury site.

Long-term In Vivo Tracking of Inflammatory Cell Dynamics Within Drosophila Pupae

Here we present a protocol for live-imaging wound repair and the associated inflammatory response at high spatio-temporal resolution in vivo. This method utilizes the pupal stage of Drosophila development to enable long-term imaging and tracking of specific cell populations over time and is compatible with efficient RNAi-mediated gene inactivation.

ショウジョウバエ蛹内炎症性細胞動態の長期的な体内の追跡

During the rapid inflammatory response to tissue damage, cells of the innate immune system are quickly recruited to the injury site. Once at the wound, ...

免疫・感染学

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent adult integument. Their immobility and transparency make them ideal for in vivo live imaging experiments. Many studies have focused on the dorsal epithelial monolayer of the pupal&#160;notum because of its accessibility and relatively large size. In addition to the studies of epithelial mechanics and development, the notum has been an ideal tissue to study wound healing. After an injury, the entire epithelial repair process can be captured by live imaging over 6-12 h. Despite the popularity of the notum for live imaging, very few published studies have utilized fixed notum samples. Fixation and staining are common approaches for nearly all other Drosophila tissues, taking advantage of the large repertoire of simple cellular stains and antibodies. However, the pupal notum is fragile and prone to curling and distortion after removal from the body, making it challenging to complement live imaging. This protocol offers a straightforward method for fixing and staining the pupal notum, both intact and after laser-wounding. With this technique, the ventral side of the pupa is glued down to a coverslip to immobilize the pupa, and the notum is carefully removed, fixed, and stained. The notum epithelium is mounted on a slide or between two coverslips to facilitate imaging from the tissue&#39;s dorsal or ventral side.

Dissecting, Fixing, and Visualizing the Drosophila Pupal Notum

The present protocol details the preparation and visualization of fixed tissue of the Drosophila pupal notum. It can be used for either intact or wounded tissue, and the original architecture of the tissue is preserved. The procedures for dissecting, fixing, and staining are all described in this article.

ショウジョウバエの蛹の解剖学的、固定、視覚化

The pupae of Drosophila melanogaster are immobile for several days during metamorphosis, during which they develop a new body with a thin transparent ...

Drosophila Larva Imaginal Disc Dissection: A Method to Observe Developing Epithelia

Source: Morimoto, K. and Tamori, Y. <a href="https://jove.unicartagenaproxy.elogim.com/video/55901/" target="_blank">Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia</a>. J. Vis. Exp. (2017).
When staining Drosophila imaginal discs, often a rough dissection is performed where the discs are exposed but remain attached to the body. Once stained, the discs of interest are isolated and mounted. The example protocol demonstrates a procedure for wing imaginal disc dissection and mounting.

ショウジョウバエ幼虫の想像上板解剖:発達中の上皮を観察する方法

Source: Morimoto, K. and Tamori, Y. Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia. J. Vis. Exp. (2017).
When staining Drosophila ...