We thank Christian Lehner for providing the CENP-C antibody and Eric Joyce for recommendations on FISH. Work in the McKim lab was funded by a grant from NIH (GM101955).

注：特に断りのない限り、手順は室温で行われます。温度制御インキュベータは、ハエの飼育のための温度を維持するために使用され、特に断りのない限り、交差しています。 1.準備<ol><li>ハエを準備します。 <ol><li>前中期に富む卵母細胞のコレクション。 <ol><li>クリア大人は、健康な、若い株式またはクロス培養から飛びます。 25℃で2日間年齢ボトル。 メモ：詳細は、いくつかのクロス文化のために必要とされるかもしれないが、一般的に2人の健康のボトルは、十分であろう。 </li><li> 2日後、ボトルから（この時点では0〜2日齢である）〜100〜300人の女性を集めます。女性が処女である必要はありません。バイアルの側に酵母ペーストのDABを追加し、yeastedバイアルに30人の女性と10〜15人の男性それぞれに配置します。 25℃で2日間年齢バイアル。 </li></ol></li><li>中期に富む卵母細胞のコレクション。 <ol><li>株式やクロスculturから約100〜300人の女性を集めますエス。バイアルの側に酵母ペーストのDABを追加し、yeastedバイアルに30人の女性（無添加の男性と）それぞれを配置します。 25℃で3〜5日間年齢バイアル。 </li></ol></li></ol></li><li>ソリューションを準備します。 注：特に断りのない限り、溶液は、室温で無期限に保存してもよいです。 <ol><li> 500mMのHEPES、500mMのスクロース、275 mM酢酸ナトリウム、200mMの酢酸カリウム、50mMのグルコース、6mMの塩化マグネシウム、および5mM塩化カルシウム：修飾ロブの緩衝液（5×）を調製します。 7.4にpHを水酸化カリウム：8水酸化ナトリウム：10 N 11を使用してください。濾過により滅菌します。オートクレーブはありません。 -20℃で保管してください。 〜1×ロブの卵母細胞あたりの準備を200mlを調製するために、必要に応じて解凍します。 </li><li>固定ソリューション。 <ol><li>オプション＃1：ホルムアルデヒド/ヘプタンの固定を準備します。 1×リン酸緩衝生理食塩水（PBS）を加えた150mMのスクロース：固定バッファを準備します。使用するには、687.5μlの固定バッファおよび312.5μlの16％ホルムアルデヒドで新鮮作ります卵母細胞の準備あたり。 注意：ドラフト内でホルムアルデヒド溶液を使用しながら、手袋を着用してください。機関のガイドラインに従って廃棄処分してください。 </li><li>オプション＃2：ホルムアルデヒド/カコジル酸固定を準備します。修正ミックスを準備：250mMのスクロース、100mMの酢酸カリウム（pHは7.5）、25 mM酢酸ナトリウム（pH 7.0）、および25mM EGTA（pHは8.0）。使用するには、400μlの新鮮なミックスを修正行い、100μlのカリウムカコジル（1 M、pHは7.2）、および卵母細胞の準備あたり500μlの16％ホルムアルデヒド。 注意：カリウムカコジルはヒ素が含まれています。 </li></ol></li><li> 1×PBS + 1％または0.05％のトリトンX-100：PBS /トリトンX-100を準備します。 4℃で保存。 </li><li> 1×PBS、（w / v）の0.5％のBSA、および0.1％のTween-20：PBS-Tween 20で、ウシ血清アルブミン（BSA）（PTB）を準備します。一週間4℃で保存してもよいです。 </li><li> in situハイブリダイゼーション（FISH）ソリューションでは 、蛍光。 <ol><li>調製20X塩化ナトリウム、クエン酸ナトリウム（SSC）：3 M塩化ナトリウムおよび0.3 Mクエン酸ナトリウムを。 </li><li>調製2×SSC、トゥイーン20（SSCT）：2×SSC + 0.1％のTween-20。卵母細胞の準備あたり新鮮な、〜20ミリリットルを加えます。 </li><li>準備ホルムアミド溶液：2×SSC、0.1％Tween-20を加えホルムアミド。 1 mlの20％ホルムアミド0.5mlの40％ホルムアミド、および卵母細胞の準備につき2 50mlの％ホルムアミド、新鮮します。 注意：ドラフト内でホルムアミド溶液を使用しながら、手袋を着用してください。機関のガイドラインに従って廃棄処分してください。 </li><li> （w / v）の2×SSC、50％ホルムアミド、10％硫酸デキストラン：ハイブリダイゼーション溶液を調製します。 4℃で保存。 </li></ol></li><li> FISHプローブ。 <ol><li>注文HPLC精製を有するオリゴヌクレオチド（配列については表1を参照）、5 &#39;蛍光修飾（ 例えば 、Cy3またはCy5の）を希望します。 50 ngの/μlのでトリスEDTA（TE）で再懸濁します。 注：すべての回で、露光からオリゴを保護します。 </li></ol></li></ol></li></ol>後期ショウジョウバエ卵母細胞の2コレクション<ol><li>として<eメートル&gt;プラスチックやガラスに固執する、無傷の膜を有するショウジョウバエの卵母細胞、プレコート1 5ミリリットルチューブとPTBと卵母細胞の準備につき1パスツールピペットの内部。 </li><li>二酸化炭素がすべて〜100〜300 yeastedハエを麻酔し、〜100ミリリットル1×ロブのバッファーを含むブレンダーに追加します。パルス3回（約1秒ずつ）。活性化を避けるために、&lt;20分間ロブの中で卵母細胞を保管してください。 注：別の方法として、卵母細胞が雌から手で解剖することができます。この方法の利点は、以下の女性を必要とすることです。しかし、ケアは、低酸素状態7に関連したアーチファクトを回避するためにわずか数分に二酸化炭素への曝露を制限するように注意する必要があります。 </li><li>大きな身体の部分を除去するために、250ミリリットルのビーカーに大きなメッシュ（〜1500ミクロン）を通して濾過。多くの無傷の腹部には、追加のロブのバッファを使用して、メッシュ、再研削材上に残り、再びフィルタする場合。その後、できるだけ大きな体の部分の多くを取り除く、最上層を吸引する、〜2分の決済をしてみましょう。 </li><li> Fiの250ミリリットルのビーカーに小さなメッシュ（〜300ミクロン）を通してLTER。追加のロブのコーティングされたパスツールピペットを用いて、第1のビーカーのうち残りの卵母細胞を洗浄します。整が〜3分してみましょう。卵母細胞が沈降します。すべてしかし〜10ミリリットルを吸引除去。 </li><li>コーティングされた5ミリリットルのチューブに収まるように10ミリリットルの限りを注ぎます。 、落ち着く液体を除去し、残りは繰り返してみましょう。追加のロブのコーティングされたパスツールピペットを用いてビーカーのうち、残りの卵母細胞を洗浄します。 〜3-5分間5ミリリットルチューブに定住してみましょう。 </li></ol> 3.薬品による治療（オプション） <ol><li>コート各卵母細胞の準備のためのPTBと第2の5 mlのチューブ。各卵母細胞分取（ 表2）のために1ミリリットルロブのそれぞれに適切な溶媒（対照）または薬剤を追加します。 </li><li>第二のコーティングされた5ミリリットルチューブに分割した卵母細胞。沈降は、液体を除去し、1チューブ及び第二のチューブに1ミリリットルロブのプラス薬物に1ミリリットルロブのプラス溶剤を追加してみましょう。薬物治療（ 表2）のための適切な時間のために章動させます。 Lらが落ち着きます。 </li></ol> 4.固定<ol><li>吸引し、オフすべての液体とすぐに1ミリリットル修正を加えます。 </li><li>オプション＃1：ホルムアルデヒド/ヘプタン固定（固定緩衝液+ 5％ホルムアルデヒド）。 <ol><li>ニューテーター上で2.5分間固定してください。 1ミリリットルのヘプタン及びボルテックス1分を追加します。 〜1分で沈降してみましょう。 </li><li>すべての液体を除去し、その後PBS 1X 1ミリリットルを追加します。ボルテックス30秒。 〜1分で沈降してみましょう。 </li><li>すべての液体を除去し、次いで1×PBSでチューブを埋めます。 注：卵母細胞は、直ちに使用するか、または室温で数時間ニューテーター上で維持することができます。 </li></ol></li><li>オプション＃2：ホルムアルデヒド/カコジル固定（ミックスプラス8％のホルムアルデヒド及び100mMのカコジルを修正）。 <ol><li>ニューテーター上で6分間固定してください。 2分の決済をしてみましょう。液体を除去し、次いで1×PBSでチューブを埋めます。 注：卵母細胞は、直ちに使用するか、または室温で数時間ニューテーター上で維持することができます。 </li></ol></li></ol> 5.取り外し膜（「ローリング」） <ol><li>コーティングされたパスツールピペットを用いて、スライドガラスのつや消し（サンドブラスト）部分に〜500〜1,000卵母細胞を追加します。鉗子を使用して、すべてのボディパーツや異物を取り除きます。卵母細胞を乾燥させないでください。必要に応じて1×PBSを追加します。 </li><li>すべての膜が除去されるまで（卵母細胞全体にカバースリップの端をドラッグすると、最も効果的に機能します。）卵母細胞およびそっと「ロール」卵母細胞の上にカバースリップを置き、定期的に必要に応じてより多くの1×PBSを追加し、顕微鏡下で進行状況を確認します。あまりにも多くの圧力が卵母細胞を破壊するように注意してください。 注：唯一の免疫蛍光のために、（または免疫蛍光なし）FISHについては、ステップ6に進み、ステップ7に進みます。 </li></ol> ショウジョウバエ卵母細胞の6抗体染色<ol><li>抽出とブロッキング<ol><li> 〜15ミリリットルのPBS / 1％トリトンX-100を含む15ミリリットルコニカルチューブに巻かれた卵母細胞を洗浄します。無未満1.5時間この溶液を超えない2時間で章動卵母細胞。このステップでは、抗体の浸透を可能にします。 </li><li>卵母細胞は、〜2分に解決しましょう。 PBS / 0.05％トリトンX-100を追加し、できるだけ多くの膜と一緒に液体を削除します。 注：膜は、圧延卵母細胞よりも遅いセトリングします。 </li><li>卵母細胞は、〜2分に解決しましょう、その後、液体のすべてが、〜1ミリリットルを削除します。転送卵母細胞を1.5 mlチューブを卒業し、残りの液体を除去します。 1時間ブロッキングし、うなだれるのために1ミリリットルPTBを追加します。 </li></ol></li><li>抗体染色<ol><li>胚に対する二次抗体の予備吸収。 注：この手順は、ショウジョウバエのタンパク質との非特異的抗体相互作用からバックグラウンド染色を排除します。 <ol><li>典型的な手順8あたりのショウジョウバエの胚（〜25μl）を収集し、固定します。 -20℃のメタノール中で保管してください。 </li><li>胚からメタノールを除去、800μlのメタノールを加えた200μlの1×PBSを追加し、15分間うなだれます。上清の500μLを削除し、500μlの1×PBSと交換。その後、15分間章動させます。 （洗浄の〜1時間の合計）を2回繰り返し、その後、PTBでフィニッシュ。 注：胚はすぐに使用するか、または室温で数時間ニューテーター上で維持することができます。 </li><li> （最終容量は300μlのであろうことを念頭に置いて、ステップ7.4を参照してください。）、液体を除去卵母細胞プレップあたり200μlにPTBで記入し、適切な希釈で蛍光標識された二次抗体を追加章動4℃で一晩（望ましいです）または3〜4時間、室温で。 注：前吸収抗体は、ステップ6.2.4で使用されます。 注：蛍光抗体が追加された後、できるだけ多くの暗闇の中でサンプルを保管してください。 </li></ol></li><li> 、卵母細胞から液体を除去する300μlにPTBで記入し、適切な希釈で一次抗体を追加します。 4℃での章動一晩（望ましい）または3〜4時間、室温で。 </li><li>洗浄は、1ミリリットルPTBで15分間、4回ずつ卵母細胞。 </li><li>削除します卵母細胞からの液体、胚（プレ吸収二次抗体）からの上清200μlを添加します。その後、300μlにPTBでいっぱい。 3〜4時間（好ましく）または4℃で一晩、室温で章動させます。 </li><li> 1ミリリットルPTBで15分間、一度卵母細胞を洗浄し、液体を除去。その後、7分間0.5μlのヘキスト33342と500μlのPTBとうなだれるを追加します。 </li><li> 1ミリリットルPTBで15分間ずつ二回卵母細胞を洗ってください。 注：卵母細胞は直ちにスライド上に取り付けられてもよいし、撮像のための準備ができるまで4℃でPTBに格納されてもよいです。 </li></ol></li></ol> 7. FISH（上記の手順5から続けます） <ol><li> 〜15ミリリットル2倍SSCTを含む15ミリリットルコニカルチューブに巻かれた卵母細胞を洗浄します。卵母細胞は、〜2分に解決しましょう。できるだけ多くの膜と一緒に液体すべてが、〜0.5ミリリットルを削除します。 注：膜は、圧延卵母細胞よりも遅いセトリングします。 </li><li>転送は、0.5 mlチューブに卵母細胞および残りの液体を除去します。続いて500μlのを追加および削除20％、40％、および50％ホルムアミドの溶液を、各溶液中に10分間章動。 注：卵母細胞は、ホルムアミドの割合が高いと遅く沈降します。 </li><li> 500μlの50％ホルムアミド溶液を加えた後、液体の削除、および1〜5時間、37℃でうなだれます。 注：インキュベーション時間が長いほど、より良いプローブの浸透につながります。 </li><li>これ以上の100〜よりμlの卵母細胞を残して、液体を除去し、36μlのハイブリダイゼーション溶液を加えたプローブの2μlの（50 ngの/μL）と水の2μlあるいは2 番目のプローブ（ 表1）の2μlを添加します。 </li><li>一晩37℃の水浴中でインキュベートし、続いて3分間（FISHのみ）または20分間（FISHプラス免疫蛍光法）のために80°C、91°Cでインキュベートします。 注：これらの手順は、サーモサイクラーで行うことができます。 </li><li>液体を除去しないでください。 500μlの1時間、37℃で50％ホルムアミド溶液と、うなだれるを追加します。 </li><li> 、液体を除去、沈降をしてみましょう500μlの50％ホルムアミド溶液を加え、1時間37℃でdはうなだれます。 </li><li>落ち着くしましょう、液体を除去、500μlの20％ホルムアミド溶液を添加し、10分間室温でうなだれます。 </li><li> 500μlの2×SSCT（沈降が、その後、液体を追加し、数回転倒、繰り返しを削除しましょう。）が沈降してみましょう、液体を除去し、4時間500μlのPTBとうなだれるを追加で3クイック回の洗浄を行います。 </li></ol> 8.抗体染色FISH後<ol><li> 、卵母細胞から液体を除去する300μlにPTBで記入し、FITCにコンジュゲート10μlの抗αチューブリン抗体を追加します。あるいは、他の抗体は、上記のプロトコルに従って、使用することができます。室温で一晩章動させます。 </li><li>その後、7分間0.5μlのヘキスト33342と500μlのPTBとうなだれるを追加し、液体を除去し、500μlのPTBとの15分間に一度卵母細胞を洗浄します。 </li><li> 500μlのPTBで15分間ずつ二回卵母細胞を洗ってください。 注：卵母細胞は直ちにスライド上に取り付けられてもよいし、準備が整うまで4℃でPTBに格納することができますイメージング。暗闇の中で卵母細胞を蛍光抗体が追加された後、可能な限りにしてください。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td fo:width="1in"> 繰り返し名前 </td><td fo:width="1in"> 染色体 </td><td> オリゴ配列* </td></tr><tr><td> 359 </td><td>バツ</td><td> GGGATCGTTAGCACTGGTAATTAGCTGC </td></tr><tr><td> AACAC </td><td> 2 </td><td> AACACAACACAACACAACACAACACAACACAACACAACAC </td></tr><tr><td>ドデカ</td><td> 3 </td><td> CCCGTACTGGTCCCGTACTCGGTCCCGTACTCGGT </td></tr><tr><td> 1.686 </td><td> 2 + 3 </td><td> AATAACATAGAATAACATAGAATAACATAG </td></tr><tr><td> AATAT </td><td> 4（+ Y） </td><td> AATATAATATAATATAATATAATATAATAT </td></tr><tr><td colspan="3"> *エリック・ジョイス、パーソナル通信、サリバンらから他の配列から359の配列。8 </td&gt; </tr></tbody></table> 表1： ショウジョウバエ 動原体繰り返し のためのFISHプローブ 。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> ドラッグ </td><td> 溶媒 </td><td> ストック濃度 </td><td> 最終濃度 </td><td> 治療の時間 </td><td> 効果 </td></tr><tr><td>コルヒチン</td><td>エタノール</td><td> 125 mMの</td><td> 150μM </td><td> 10分または30分</td><td>非動原体（10分）9またはすべて（30分）微小管を不安定にします</td></tr><tr><td>パクリタキセル</td><td> DMSO </td><td> 10 mMの</td><td> 10μM </td><td> 10分</td><td>マイクを安定させますrotubules </td></tr><tr><td> Binucleine 2 </td><td> DMSO </td><td> 25 mMの</td><td> 25μM </td><td> 20分</td><td>オーロラBキナーゼ10を阻害</td></tr></tbody></table> 表2：薬物治療。

<ol>
	<li>Bridges, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-disjunction+as+proof+of+the+chromosome+theory+of+heredity.">Non-disjunction as proof of the chromosome theory of heredity.</a> Genetics. 1 (1), 1-52 (1916).</li><li>Hassold, T., Hunt, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=To+err+(meiotically)+is+human:+the+genesis+of+human+aneuploidy.">To err (meiotically) is human: the genesis of human aneuploidy.</a> Nat Rev Genet. 2 (4), 280-291 (2001).</li><li>Theurkauf, W. E., 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=Meiotic+spindle+assembly+in+Drosophila+females:+behavior+of+nonexchange+chromosomes+and+the+effects+of+mutations+in+the+nod+kinesin-like+protein.">Meiotic spindle assembly in Drosophila females: behavior of nonexchange chromosomes and the effects of mutations in the nod kinesin-like protein.</a> J Cell Biol. 116 (5), 1167-1180 (1992).</li><li>Zou, J., Hallen, M. A., Yankel, C. D., Endow, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microtubule-destabilizing+kinesin+motor+regulates+spindle+length+and+anchoring+in+oocytes.">A microtubule-destabilizing kinesin motor regulates spindle length and anchoring in oocytes.</a> J Cell Biol. 180 (3), 459-466 (2008).</li><li>Dernburg, A. F., Sedat, J. W., 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=Direct+evidence+of+a+role+for+heterochromatin+in+meiotic+chromosome+segregation.">Direct evidence of a role for heterochromatin in meiotic chromosome segregation.</a> Cell. 86 (1), 135-146 (1996).</li><li>Gilliland, W. D., Hughes, S. F., Vietti, D. R., 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=Congression+of+achiasmate+chromosomes+to+the+metaphase+plate+in+Drosophila+melanogaster+oocytes.">Congression of achiasmate chromosomes to the metaphase plate in Drosophila melanogaster oocytes.</a> Dev Biol. 325 (1), 122-128 (2009).</li><li>Gilliland, W. D., 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=Hypoxia+transiently+sequesters+mps1+and+polo+to+collagenase-sensitive+filaments+in+Drosophila+prometaphase+oocytes.">Hypoxia transiently sequesters mps1 and polo to collagenase-sensitive filaments in Drosophila prometaphase oocytes.</a> PLoS One. 4 (10), e7544 (2009).</li><li>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=.">.</a> Drosophila Protocols. , (2000).</li><li>Radford, S. J., Hoang, T. L., G&#322;uszek, A. A., Ohkura, H., McKim, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lateral+and+End-On+Kinetochore+Attachments+Are+Coordinated+to+Achieve+Bi-orientation+in+Drosophila+Oocytes.">Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes.</a> PLoS Genet. 11 (10), e1005605 (2015).</li><li>Smurnyy, Y., Toms, A. V., Hickson, G. R., Eck, M. J., Eggert, U. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binucleine+2,+an+isoform-specific+inhibitor+of+Drosophila+Aurora+B+kinase,+provides+insights+into+the+mechanism+of+cytokinesis.">Binucleine 2, an isoform-specific inhibitor of Drosophila Aurora B kinase, provides insights into the mechanism of cytokinesis.</a> ACS Chem Biol. 5 (11), 1015-1020 (2010).</li><li>Mahowald, A. P., Goralski, T. J., Caulton, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+activation+of+Drosophila+eggs.">In vitro activation of Drosophila eggs.</a> Dev Biol. 98 (2), 437-445 (1983).</li><li>Page, A. W., Orr-Weaver, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+the+meiotic+divisions+in+Drosophila+oocytes.">Activation of the meiotic divisions in Drosophila oocytes.</a> Dev Biol. 183 (2), 195-207 (1997).</li><li>Tavosanis, G., Llamazares, S., Goulielmos, G., Gonzalez, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+role+for+gamma-tubulin+in+the+acentriolar+female+meiotic+spindle+of+Drosophila.">Essential role for gamma-tubulin in the acentriolar female meiotic spindle of Drosophila.</a> EMBO J. 16 (8), 1809-1819 (1997).</li><li>Endow, S. A., Komma, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spindle+dynamics+during+meiosis+in+Drosophila+oocytes.">Spindle dynamics during meiosis in Drosophila oocytes.</a> J Cell Biol. 137 (6), 1321-1336 (1997).</li><li>Matthies, H. J., Clarkson, M., Saint, R. B., Namba, R., Hawley, R. S., 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=.">.</a> Drosophila Protocols. , 67-85 (2000).</li><li>Colombi&#233;, N., 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=Dual+roles+of+Incenp+crucial+to+the+assembly+of+the+acentrosomal+metaphase+spindle+in+female+meiosis.">Dual roles of Incenp crucial to the assembly of the acentrosomal metaphase spindle in female meiosis.</a> Development. 135 (19), 3239-3246 (2008).</li></ol>

The authors have nothing to disclose.

ショウジョウバエの卵母細胞ステージング 細長いカリオソームは、多くの場合、中期卵母細胞から前中期区別するためにカリオソーム形状を使用して、前中期卵母細胞に見られるが問題となる可能性があります。前中期の間、カリオソームは、丸い形状として始まり伸長し、その後卵母細胞が中期停止に近づくにつれて円形に後退します。これは、?...

The study of meiosis is sometimes described as the "genetics of genetics". This is because the fundamental properties of chromosome inheritance and independent assortment are carried out through the segregation of chromosomes during gamete production. An important demonstration of the chromosome theory of inheritance came in 1916 from the work of Calvin Bridges in Drosophila melanogaster1. This and other classical genetics studies in Drosophila contributed greatly to our understanding of genetics. Cytological examination of meiotic chromosomes in Drosophila oocytes, however, has been challenging. This is primarily because immunofluorescence of late-stage Drosophila oocytes, when the spindle assembles and chromosomes are oriented for segregation, is hampered by the presence of membranes that render the oocyte impenetrable to antibodies.
Despite this challenge, Drosophila oocytes remain an attractive model for the study of chromosome behavior and spindle assembly. This is because of the powerful genetic tools available in Drosophila, but also because the oocytes arrest at metaphase I, when the chromosomes are oriented and the spindle is fully formed. This facilitates the collection and examination of large numbers of oocytes at this important stage of cell division. In addition, a simple model organism that is amenable to genetic manipulation for the study of oocyte chromosome segregation can provide an important contribution to our understanding of human reproductive health. Errors in chromosome number are the leading genetic cause of miscarriage and birth defects in humans2. A majority of these errors can be traced to the oocyte and are correlated with increasing maternal age. The average age of mothers in the U.S. has been increasing, making this a major public health concern.
We describe here methods for the cytological examination of Drosophila oocytes, including a demonstration of how to remove the oocyte membranes. These methods are modifications of protocols first described by Theurkauf and Hawley3, Zou et al.4, and Dernburg et al.5. We also include methods for the enrichment of different stages of oocytes, based on a protocol first described by Gilliland et al.6. Finally, we add instructions for the drug treatment of Drosophila oocytes. Together, these methods allow the cytological investigation of oocyte chromosome segregation and spindle assembly in Drosophila.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>15 mL conical tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>16% formaldehyde</td>
			<td>Ted Pella, Inc.</td>
			<td>18505</td>
			<td>HAZARDOUS; once opened, discard after one month</td>
		</tr>
		<tr>
			<td>250 mL beakers</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>active dry yeast</td>
			<td>Various</td>
			<td></td>
			<td>mix with water to make a paste the consistency of peanut butter</td>
		</tr>
		<tr>
			<td>anti-&#945;-tubulin antibody conjugated to FITC</td>
			<td>Sigma</td>
			<td>F2168</td>
			<td>clone DM1A</td>
		</tr>
		<tr>
			<td>Binucleine 2</td>
			<td>Sigma</td>
			<td>B1186</td>
			<td>HAZARDOUS</td>
		</tr>
		<tr>
			<td>blender</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>bovine serum albumin</td>
			<td>Sigma</td>
			<td>A4161</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chloride</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>colchicine</td>
			<td>Sigma</td>
			<td>C-9754</td>
			<td>HAZARDOUS</td>
		</tr>
		<tr>
			<td>coverslips</td>
			<td>VWR</td>
			<td>48366-227</td>
			<td>No. 1 1/2</td>
		</tr>
		<tr>
			<td>dextran sulfate</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Ted Pella, Inc.</td>
			<td>5622</td>
			<td>Dumont tweezers high precision grade style 5</td>
		</tr>
		<tr>
			<td>formamide</td>
			<td>Sigma</td>
			<td>47670-250ML-F</td>
			<td></td>
		</tr>
		<tr>
			<td>glass slides</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td></td>
		</tr>
		<tr>
			<td>glucose</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>graduated 1.5 mL tubes</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>VWR</td>
			<td>EM-5330</td>
			<td>available from several venders</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium chloride</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>large mesh (~1500 &#181;m)</td>
			<td>VWR</td>
			<td>AA43657-NK</td>
			<td>variety of formats and other suppliers, 12 or 14 mesh</td>
		</tr>
		<tr>
			<td>small mesh (~300 &#181;m)</td>
			<td>Spectrum labs</td>
			<td>146 424</td>
			<td>variety of formats, eg 146 422 or 146 486</td>
		</tr>
		<tr>
			<td>nutator</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipets</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>potassium acetate</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cacodylic acid</td>
			<td>Sigma</td>
			<td>C0125</td>
			<td>HAZARDOUS; alternatively, sodium cacodylate may be substituted</td>
		</tr>
		<tr>
			<td>potassium hydroxide</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium acetate</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium citrate</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>taxol (paclitaxel)</td>
			<td>Sigma</td>
			<td>T1912</td>
			<td>HAZARDOUS</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher</td>
			<td>PI-28314</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Fisher</td>
			<td>PI-28320</td>
			<td></td>
		</tr>
		<tr>
			<td>vortex</td>
			<td>Various</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

techniques for imaging prometaphase and metaphase of meiosis i in fixed drosophila oocytes

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The study of chromosome behavior in oocytes from model organisms holds much promise to uncover the molecular basis of the susceptibility of human oocytes to aneuploidy. Drosophila melanogaster is amenable to genetic manipulation, with over 100 years of research, community, and technique development. Visualizing chromosome behavior and spindle assembly in Drosophila oocytes has particular challenges, however, due primarily to the presence of membranes surrounding the oocyte that are impenetrable to antibodies. We describe here protocols for the collection, preparation, and imaging of meiosis I spindle assembly and chromosome behavior in Drosophila oocytes, which allow the molecular dissection of chromosome segregation in this important model organism.

我々がここで説明している方法は、減数分裂の三段階を表す後期ショウジョウバエ卵母細胞の収集（ 図1）になります。前期中の卵母細胞はカリオソーム（ 図1A）を取り囲む領域におけるチューブリンシグナルの欠如によって可視である、核膜の存在によって区別されます。前中期は、核膜の崩壊時のスピンドル組み立て後の期間です?...

Watch this Scientific Journal Video about Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes at JoVE.com

Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

固定における減数分裂Iのイメージング前中期および中期のための技術<em&gt;ショウジョウバエ</em&gt;卵母細胞

Chromosome segregation in human oocytes is error prone, resulting in aneuploidy, which is the leading genetic cause of miscarriage and birth defects. The ...

techniques-for-imaging-prometaphase-metaphase-meiosis-i-fixed

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Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Unit 2

Meiosis

SCIENTISTS IN ACTION

9.0K ビュー.  Rutgers University. We present protocols for the collection, preparation, and imaging of mature Drosophila oocytes. These methods allow the visualization of chromosome behavior and spindle assembly and function during meiosis.

ビデオ: Techniques for Imaging Prometaphase and Metaphase of Meiosis I in Fixed Drosophila Oocytes

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

に有糸分裂研究のためのマイクロインジェクション技術キイロショウジョウバエ合胞体胚

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

生物学

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 Developing and Adult Drosophila Brains and Retinae for Live Imaging

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three preparations of the brain and visual system that cover the range from the developing eye disc-brain complex in the developing pupae to individual eye and brain dissection from adult flies. All protocols are optimized for the live culture of the preparations. However, we also present the conditions for fixed tissue immunohistochemistry where applicable. Finally, we show live imaging conditions for these preparations using conventional and resonant 4D confocal live imaging in a perfusion chamber. Together, these protocols provide a basis for live imaging on different time scales ranging from functional intracellular assays on the scale of minutes to developmental or degenerative processes on the scale of many hours.

Preparation of Developing and Adult Drosophila Brains and Retinae for Live Imaging

This protocol describes three Drosophila preparations: 1) adult brain dissection, 2) adult retina dissection and 3) developing eye disc- brain complexes dissection. Emphasis is laid on special preparation techniques and conditions for live imaging, although all preparations can be used for fixed tissue immunohistochemistry.

開発と大人の準備ショウジョウバエ</emライブイメージングのための>脳と網膜

The Drosophila brain and visual system are widely utilized model systems to study neuronal development, function and degeneration. Here we show three ...

Techniques for Imaging Ca2+ Signaling in Human Sperm

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in live cells. Here we describe the method used in our laboratories for loading suspensions of human sperm with Ca2+-reporting dyes and measuring the fluorescence signal during physiological stimulation. Motile cells are isolated by direct swim-up and incubated under capacitating conditions for 0-24 h, depending upon the experiment. The cell-permeant AM (acetoxy methyl ester) ester form of the Ca2+-reporting dye is then added to a cell aliquot and a period of 1 h is allowed for loading of the dye into the cytoplasm. We use visible wavelength dyes to minimize photo-damage to the cells, but this means that ratiometric recording is not possible. Advantages and disadvantages of this approach are discussed. During the loading period cells are introduced into an imaging chamber and allowed to adhere to a poly-D-lysine coated coverslip. At the end of the loading period excess dye and loose cells are removed by connection of the chamber to the perfusion apparatus. The chamber is perfused continuously, stimuli and modified salines are then added to the perfusion header. Experiments are recorded by time-lapse acquisition of fluorescence images and analyzed in detail offline, by manually drawing regions of interest. Data are normalized to pre-stimulus levels such that, for each cell (or part of a cell), a graph showing the Ca2+ response as % change in fluorescence is obtained.

Techniques for Imaging Ca2+ Signaling in Human Sperm

Stimulus-evoked [Ca2+]i signals of individual human sperm are assessed. Motile cells are loaded with Ca2+-sensitive fluorescent dye (AM-ester method) and immobilised in a perfusable chamber. Cells are imaged by time-lapse fluorescence microscopy and stimulated via the perfusing medium. Responses of single cells (or regions) are analysed offline using Excel.

イメージングカルシウムのためのテクニック 2 +人間の精子でシグナリング

Fluorescence microscopy of cells loaded with fluorescent, Ca2+-sensitive dyes is used for measurement of spatial and temporal aspects of Ca2+ signaling in ...

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 ...

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 ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

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 ...

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

The Drosophila oocyte has been established as a versatile system for investigating fundamental questions such as cytoskeletal function, cell organization, and organelle structure and function. The availability of various GFP-tagged proteins means that many cellular processes can be monitored in living cells over the course of minutes or hours, and using this technique, processes such as RNP transport, epithelial morphogenesis, and tissue remodeling have been described in great detail in Drosophila oocytes1,2.
The ability to perform video imaging combined with a rich repertoire of mutants allows an enormous variety of genes and processes to be examined in incredible detail. One such example is the process of ooplasmic streaming, which initiates at mid-oogenesis3,4. This vigorous movement of cytoplasmic vesicles is microtubule and kinesin-dependent5 and provides a useful system for investigating cytoskeleton function at these stages.
Here I present a protocol for time lapse imaging of living oocytes using virtually any confocal microscopy setup.

Live Imaging of GFP-labeled Proteins in Drosophila Oocytes

A protocol for live imaging of GFP-tagged proteins or autofluorescent structures in individual Drosophila oocytes is described.

でGFP標識タンパク質のイメージングを生きる<em&gt;ショウジョウバエ</em&gt;卵母細胞

The  Drosophila oocyte has been established as a versatile system for investigating  fundamental questions such as cytoskeletal function, cell ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

アポトーシス細胞のクリアランスのイメージング中に生きる<em&gt;ショウジョウバエ</em&gt;胚

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

固定における減数分裂Iのイメージング前中期および中期のための技術

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開発と大人の準備ショウジョウバエ脳と網膜

イメージングカルシウムのためのテクニック^{2 +}人間の精子でシグナリング

の直立イメージングショウジョウバエ胚

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

イオンチャネル研究のためのパッチクランプおよび灌流のテクニックで表さアフリカツメガエル卵母細胞

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

でGFP標識タンパク質のイメージングを生きる

アポトーシス細胞のクリアランスのイメージング中に生きる