We appreciate assistance from members of the fly community, particularly Dr. Denise Montell, Dr. Lynn Cooley, and Dr. Pernille Rorth, for reagents. We thank Flybase, the Bloomington Drosophila Stock Center, and Developmental Studies Hybridoma Bank for information and providing fly stocks and antibodies, respectively. LM is supported by the Department of Education&#160;Grant, Graduate Assistance in the Areas of National Need (GAANN) training fellowship (P200A120017) and by a NIGMS Initiative for Maximizing Student Development Grant (2 R25-GM55036). A portion of the microscopy work was supported by NSF MRI grant DBI-0722569 and the Keith R. Porter Core Imaging Facility. Research was supported in part by a NSF CAREER Award (1054422) and a Basil O&#8217;Connor Starter Scholar Award from the March of Dimes, both awarded to MSG.

Ovariolesの1。解剖<ol><li>追加された活性乾燥酵母を新鮮なフライ食品のバイアルに約15 2-4日齢の雌のハエや少数の男性を転送します。バイアル用のプラグとして綿を使用し、高湿度を保つために綿に水を数滴を追加します。 </li><li>段8-10卵室の数を最大にするために14〜16時間、25℃のインキュベーター内の場所バイアル。 注：インキュベーション時間は、温度および所望の段階に応じて変化する。 </li><li>次の日のために、必要に応じて解剖メディアを準備し、0.1M KPO 4とNP40ソリューションは、（材料を参照）。 </li><li> CO 2を使用してハエを麻酔し、解剖顕微鏡下に置く。ピペットガラスうつ病スライド内の各キャビティ内に解剖メディアの数滴。 </li><li>一人の女性フライダウン翼を持つテーブルトップと東洋へ約30度の角度でそれぞれの手で鉗子を持ちます。あなたの非利き手では、Aで彼女を把握し、ピンセットを用いて、女性を拾う胸部の近くに腹部のnterior、およびうつ病のスライド（ 図1Aの挿入図）で解剖メディアに彼女を沈める。 </li><li>鉗子の他のペアで、腹部の腹側後部に外骨格を把握し、を貫通。後部端の卵巣をつかみ、その後腹部からそれらを引き出し、うつ病のスライドの反対側に新鮮な培地に入れる。 （ 図1Aの挿入図）。腸を引き離すことは避けてください。死体を捨てる。 </li><li>すべての卵巣を解剖されるまで、他の女性のハエで繰り返します。男性を捨てる。 </li><li>鉗子の1対の、（最大の卵室の近く）の後方端で卵巣を保持し、もう一方の手で最も前方の卵室（germarium）の1を挟ま鉗子のペアを使用。 （ 図1A）。 </li><li>ゆっくりと着実に、卵巣シースの外に卵巣小管チェーンを引っ張る。すべての目的の卵室がRになるまで個別にovariolesを解剖していきemoved。 </li><li>繰り返しますが、すべての解剖卵巣のための1.7から1.8を繰り返します。 </li><li>完了したら、プラスチック製のトランスファーピペットを用いて、0.6ミリリットルチューブにovariolesを移す。 </li><li> ovariolesは、チューブの底に沈殿、定着し、染色のステップに進みましょう。 </li></ol> 2.免疫蛍光（IF）Ovariolesの染色<ol><li>ピペットで注意深くovariolesから解剖メディアを削除する。すべてのovariolesまたは卵室を削除しないように注意してください。 ovariolesと解剖メディアの約50μlのままにしておきます。 </li><li>チューブ中の0.1M KPO 4緩衝液150μlの16％パラホルムアルデヒドの50μlのを追加し、それらを修正するために卵室にソリューションを追加します。 10分間ロッキングしながらインキュベートする。注意：ホルムアルデヒドは有毒である。 </li><li>固定液を除去し、0.5ミリリットルNP40の洗浄緩衝液で2回すすぐ。 </li><li> RTで15分間ずつ2回洗浄する。 </li><li>その後の工程のために、標準的なIFプロトコル15を参照してください。 <ol><li>簡単に言えば、固定後に、追加適切な希釈で一次抗体と4℃でO / Nインキュベートする。 （：200希釈液1）二次抗体を追加し、NP40で数回洗浄する。 </li><li> NP40中で数回洗浄し、その後DAPI（1：1,000）を追加し、10分間、その後の洗浄を繰り返します。プロトコルが完了したIF回、卵室にPBS中50％グリセロールの200μlを添加し、室温で2時間放置。 </li><li> 、溶液を除去し、PBS中70％のグリセロールと交換し、そしてホイルでカバーしています。取り付けの準備が整うまで4ᵒCでサンプルを置きます。一方、ステップ3に進みます。 </li></ol></li></ol>グリセリンゼリースライド3.事前準備<ol><li>スパチュラを使用してゼリーグリセリンの少なくとも4 mlで測定し、ハーフウェイマークにガラス細胞培養管を埋める。 30分間55℃の水浴中でグリセリンゼリーを溶かす。 </li><li> 2顕微鏡スライド上に原液からのグリセロールの小滴、約300μLを、注ぐ。組織を使用して、薄い1aにグリセロールを広めるスライドの各々にわたるヤー。これは、ベースを提供し、ステップ5.6で、グリセリンゼリーを容易に除去することができます。 </li><li>フィルタリング千μlのピペットチップのオフ先端をカット。各顕微鏡スライドにゼリーゆっくり暖かいグリセリンの1,000〜1,500μLを転送するために使用するカットピペットチップ。バブルを防ぐために注意してください。 </li><li>グリセリンゼリースライドを設定するために、室温で5分間座ってみましょう。 </li><li> 60ミリメートル×15ミリメートルのペトリ皿内の各グリセリンゼリースライドを配置し、ラップでカバーしています。 4℃CO / Nにおける場所。必要に応じて、ストアは4℃で最長5日間ゼリースライドグリセリン。 </li></ol> 4.取付エッグチェンバース<ol><li>ステップ2.5、1グリセリンゼリースライド全体で70％のグリセロールで卵室のピペットいくつかの3μlの滴からのサンプルを使用して。すべての卵室は、このスライド上になるまでピペッティング3μlの滴を続行。各ドロップは、少なくとも10卵室が含まれていることを確認してください。一方、55℃の水浴中でグリセリンゼリーの培養管を加熱する。 </li><li>解剖顕微鏡下で卵室とグリセリンゼリースライドを置きます。卵室のみ一滴の視野（ 図1B）になるように倍率を調整する。 </li><li>スプーンのように30ゲージの針を用いて、所望の段階の卵室を拾う。必要な場合には、ナイフなどの針を用いて卵巣小管チェーンから目的の卵室を分離する。 </li><li>イメージングに干渉する可能性デブリを回避するために、前方（ 図1E -1）左に向けて、第グリセリンゼリースライドに卵室を転送する。少なくとも7つの卵室があるまで繰り返し列（ 図1C）に並んだ。 </li><li>すべての目的の卵室は第二グリセリンゼリースライドに転送されるまで、7の新しい列を形成する。 </li><li>組織とねじれの小片を切り取り。軽くそれぞれ1に触れて、卵室から過剰のPBS-グリセロールを吸収するために使用します。卵室を削除しないように注意してください。 </li><li>断つフィルタリング200μlのピペットチップの先端。卵室のすべての列をカバーするために、グリセリンゼリー150μlのを転送するために使用します。 注：列をカバーするのに必要なグリセリンゼリーの量は卵室段と列の数に基づいている。量を調整することができる。 </li><li>グリセリンゼリーのトップ層が完全に（ 図1D）固化するまで取り付けられた卵室は室温で5分間座ってみましょう。 </li><li>置き60ミリメートル×15ミリメートルのペトリ皿に搭載された卵室のスライドとは、ラップでカバーしています。 4℃CO / Nでストア（光退色が懸念される場合には、暗所で行わ）グリセリンゼリー層が一緒に固化することを可能にする。 </li></ol>イメージング用エッグチェンバースの5準備<ol><li>解剖顕微鏡下に取り付けられた卵室のスライドを置きます。卵室の1列のみが視野に入るように倍率を調整する。 </li><li> 45°の角度ミニチュアメスを用いて、straigをカット（卵室の後側に近い）卵室の最初の列の右側に沿ってhtのライン。 （ 図1D-E） </li><li>次に、列の最後の卵室上部の最初の卵室の上下にカット。この時点で、卵室の列には、三方をカットする必要があります。 </li><li>卵室（ 図1E -3）の前方側にできる限り近いマイクロナイフ、列の左側に沿ってスライスし、使用。 </li><li>カットカラムに冷たい1X PBT100μlのピペット。 </li><li>スライド上の残りの部分を残して、卵室のカット列の三辺の周りに余分なグリセリンゼリーを削除し、破棄するように丸い刃へらを使用してください。 </li><li>卵室の前方側で行われたカットでへらを挿入し、主要グリセリンゼリーブロックから列を分離する。 </li><li>反転した（5.8.1）または直立のどちらかのためのサンプル（5.8.2）顕微鏡を準備します。 <ol><li>倒立顕微鏡用：転送トン卵室の前方側を下に向けてカバースリップに卵室の彼が列。グリセリンゼリーのすべての列を削除し、マウントされるまで繰り返します5.2から5.8を繰り返します。乾燥からそれらを防ぐために卵室の各ブロックの上に50％のPBS-グリセロールの滴のカップルを追加します。直接カバースリップ上の画像卵室。 </li><li>正立顕微鏡の場合：前方側を上にして顕微鏡スライドに卵室の列を転送します。グリセリンゼリーのすべての列が削除され、顕微鏡スライドにマウントされるまで繰り返します5.2から5.9を繰り返します。 ＃1 1/2カバーガラスで卵室とカバーブロックの各ブロックの上に50％のPBS-グリセロールの滴のカップルを追加します。 </li></ol></li></ol>

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The authors have no competing financial interests.

ここでは、エンド·オン視点から卵室を開発し、マウントする方法を説明し、画像小。イメージング卵室のための一般的な技術は、横方向の景色のために最適化し、蛍光抗体で染色したときに主に内外方向包細胞の正確な可視化を可能にしている。複数の焦点面を見るのZ-スタックまたは3次元再構成助剤の使用が、それでも楕円形の卵室（ 図2D）の極の細胞内解像度には不十分で...

キイロショウジョウバエの研究は、現象の広い範囲の遺伝的調節に大きな洞察を提供してきました。卵形成は、組織パターニング、細胞極性の変化、細胞周期の切り替え、および翻訳調節1,2,3,4を含む多くの異なる発生プロセスを調査する扱いやすい方法を提供するので、特に、卵の開発に関する広範な研究があった。卵形成時の重要な形態形成のイベントは（5で検討）ボーダー細胞と呼ばれる細胞のセットの仕様、運動性の獲得、および移行である。細胞移動するので、動物の形態形成の重要な特徴であり、このプロセスの遺伝的調節は、よく保存されているため、ハエで決定メカニズムは、他の文脈において重要である可能性が高い。従って、我々は国境細胞遊走の分子制御を研究している。私たちの研究のために、我々は、発展途上の卵の前極を観察する新しい方法を開発した、ここで、境界セルは、それらが詳細に開発かを検討するために、発生する。 卵巣内では、様々な発生段階の卵室が薄いシース（ 図1A）に収められていovariolesと呼ばれるチェーンに沿って存在する。各卵室は、1個の卵を形成するために移動します。卵形成の間に、いくつかの異なる細胞型が協調して開発しなければならない。 D.ショウジョウバエの卵室は、単層濾胞上皮6に囲まれた1卵母細胞を含む16生殖細胞から成る。特殊化した細胞の小さな数は、上皮の前部および後部の極で発生する、極性細胞と呼ばれる。 6-8ボーダー細胞は極性細胞5,7により誘導された卵室の前方上皮、に由来する。中期卵形成（ステージ9）では、ボーダー細胞は、その隣人から切り離し、卵室5,7の後部に卵母細胞に到達するために看護師セル間で移動する。この移動は、達成されなければならない境界セルは、この集団の細胞遊走の種類て、2つの非運動性極性細胞の周囲のクラスタに残っている。境界セルクラスタの移行を成功さは受精のために必要がある、卵の殻の卵門の適切な発展を保証します。 前極細胞は、シグナル伝達カスケードを活性化することにより、境界細胞の運命を指示する。ポーラー細胞は卵母細胞の発達8,9の段階8の間に包細胞を、隣接上、Domeless（DOME）、膜貫通受容体に結合するサイトカイン、不対（UPD）を、分泌する。 UPDの結合は、転写のシグナルトランスデューサーおよび活性化因子（STAT）8,10,11をリン酸化するヤヌスチロシンキナーゼ（JAK）を引き起こす。 STATはその後転写を活性化するために、核に移動します。遅い境界細胞（SLBO）STATの直接的な転写ターゲットであり、また、境界細胞遊走12に必要とされる転写因子である。卵室の側面図は、Tを示している帽子STAT活性は、前方上皮8,11,13-全体の勾配で規制されている。極性細胞に最も近い包細胞は、このように、彼らは国境細胞になると、隣接する生殖系列の組織に侵入、活性化したSTATの最高レベルを持っている。 ボーダー細胞が内で指定されているかを理解し、上皮から切り離すために、我々は組織が編成されているかを観察する必要があります。我々は、前·オン視点から卵室を表示する場合は、極性細胞周囲の毛包細胞中のSTAT活動の放射状の対称性を期待する。エンド上のビューは、より正確に前と異なる焦点面で細胞を比較するより剥離時の膜タンパク質と細胞間のインタフェースの違いを示すであろう。卵室が楕円形と茎細胞によって互いに取り付けられているので、それらが困難前方アーキテクチャを観察すること、横方向にスライド上に沈降する。このように、卵室の極にある細胞に関する多くの情報があります側面図から推測されて。一部の情報は、光退色光セクション、光散乱、のアルゴリズムの3-D再構成、及びZ軸方向の分解能の悪い限界を介して取得することができるが、超解像顕微鏡14のような高価な技術が存在しない場合、この情報は、以下の詳細および信頼性を高める。セクションベースのイメージング（例えば、電子顕微鏡またはミクロトーム切片）他の種類のアーチファクトのために可能性を増加させる、脱水を含む組織の大規模な操作を必要とする。従って、我々は画像Dに新しい方法を開発キイロ卵室直立している。この方法は、すでに（ 審査中で、代表的な結果とマニングらを参照）運動性の細胞が運命づけられているかの解明に有用であることが証明され、卵形成の他の側面の研究でより広く貴重である可能性が高いしている。

<table><tbody><tr><td>0.1 M Potassium phosphate Buffer (KPO&lt;sub&gt;4&lt;/sub&gt; Buffer)</td><td></td><td></td><td>Add 3.1 g of NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&amp;bull;H&lt;sub&gt;2&lt;/sub&gt;O and 10.9 g of Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; (anhydrous) to distilled H&lt;sub&gt;2&lt;/sub&gt;O to make a volume of 1 L. The pH of the final solution will be 7.4. This buffer can be stored for up to 1 month at 4&amp;deg;C.</td></tr><tr><td>16% Paraformaldehyde aqueous solution, EM grade</td><td>Electron Microscopy Sciences</td><td>15710</td><td>methanol-free to preserve GFP fluorescence</td></tr><tr><td>30G x 1/2 inch needle&amp;nbsp;</td><td>VWR</td><td>BD305106</td><td>Regular bevel</td></tr><tr><td>Active Dry Yeast</td><td>Genesee Scientific</td><td>62-103</td><td>To fatten female flies</td></tr><tr><td>Bovine Serum Albumin</td><td>PAA-cell culture company</td><td>A15-701</td><td>Used in NP40 Wash Buffer</td></tr><tr><td>Dumont #5 Forceps</td><td>Fine Science Tools</td><td>11295-10</td><td>Dumostar alloy, biologie tip; sharp tips are essential for ovariole dissection</td></tr><tr><td>DAPI (4&amp;prime;,6-Diamidino-2-phenylindole dihydrochloride)</td><td>Sigma Aldrich</td><td>D9542</td><td>5 mg/ml stock solution&amp;nbsp;&amp;nbsp;</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16140-071</td><td>Added to supplement dissection medium (10%)&amp;nbsp;</td></tr><tr><td>Glass culture tube</td><td>VWR</td><td>47729-576</td><td>14 ml</td></tr><tr><td>Glass Depression Slides</td><td>VWR</td><td>470019-020</td><td>1.2 mm Thick, Double Cavity</td></tr><tr><td>Glycerin Jelly&amp;nbsp;</td><td>Electron Microsocpy Sciences</td><td>17998-10</td><td>Mounting media</td></tr><tr><td>Glycerol</td><td>IBI Scientific</td><td>IB15760</td><td>70% in PBS</td></tr><tr><td>IGEPAL CA-630</td><td>Sigma Aldrich</td><td>I3021-500ML</td><td>(interchangable for Nonidet P-40)&amp;nbsp; Used in NP40 Wash Buffer&amp;nbsp;</td></tr><tr><td>Leica Fluorescent Stereoscope&amp;nbsp;</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Leica SP5 Confocal Microscope</td><td>Leica Microsystems</td><td></td><td>40x/0.55NA dry objective&amp;nbsp;</td></tr><tr><td>Micro spatula&amp;nbsp;</td><td>VWR</td><td>82027-518</td><td>Stainless steel</td></tr><tr><td>Microscope Slide</td><td>VWR</td><td>16004-368</td><td>75x25x1 mm</td></tr><tr><td>NP40 Wash Buffer</td><td></td><td></td><td>50 mM TRIS-HCl, 150 mM NaCl, 0.5% Ipegal, 1 mg/ml BSA, and 0.02% sodium azide</td></tr><tr><td>Penicillin-Streptomycin-Glutamine</td><td>Life Technologies</td><td>10378-016</td><td>Added to supplement dissection medium (0.6X)</td></tr><tr><td>Petridish- Polysterine, sterile</td><td>VWR</td><td>82050-548</td><td>60 W x 15 H mm</td></tr><tr><td>Phosphate Buffer Saline</td><td>Sigma Aldrich</td><td>P3813-10PAK</td><td>10 packs of Powder</td></tr><tr><td>Potassium phosphate dibasic</td><td>Sigma Aldrich</td><td>P3786-500G</td><td>Used in 0.1 M KPO&lt;sub&gt;4&lt;/sub&gt; Buffer&amp;nbsp;</td></tr><tr><td>Potassium phosphate monobasic</td><td>Sigma Aldrich</td><td>P9791-500G</td><td>Used in 0.1 M KPO&lt;sub&gt;4&lt;/sub&gt; Buffer&amp;nbsp;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Schneider&amp;rsquo;s Insect Medium</td><td>Life Technologies</td><td>&lt;br/&gt; 11720018</td><td>With L-glutamine and sodium bicarbonate</td></tr><tr><td>Sodium azide</td><td>Sigma Aldrich</td><td>S2002-25G</td><td>Used in fluorescent antibody staining</td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>S3014-500G</td><td>Used in NP40 Wash Buffer</td></tr><tr><td>TRIS-HCl</td><td>IBI Scientific</td><td>IB70144</td><td>1 M TRIS-HCl, pH 7.4</td></tr><tr><td>Volocity 3D Image Analysis Software</td><td>PerkinElmer</td><td></td><td>For processing confocal Z-stacks</td></tr></tbody></table>

upright imaging of drosophila egg chambers

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in architecture, is well-characterized, and is amenable to genetic analysis. Each egg chamber consists of germ-line cells surrounded by a single epithelial layer of somatic follicle cells. Subsets of follicle cells undergo differentiation during specific stages to become several different cell types. Standard techniques primarily allow for a lateral view of egg chambers, and therefore a limited view of follicle cell organization and identity. The upright imaging protocol describes a mounting technique that enables a novel, vertical view of egg chambers with a standard confocal microscope. Samples are first mounted between two layers of glycerin jelly in a lateral (horizontal) position on a glass microscope slide. The jelly with encased egg chambers is then cut into blocks, transferred to a coverslip, and flipped to position egg chambers upright. Mounted egg chambers can be imaged on either an upright or an inverted confocal microscope. This technique enables the study of follicle cell specification, organization, molecular markers, and egg development with new detail and from a new perspective.

直立イメージング法は、私たちはステージ8の前方濾胞上皮に直接卵胞細胞の運命のための一般的なマーカーを細胞の組織を見ることができ、目の不在（EYA）タンパク質だけでなく、核DNAマーカーDAPIは、さえ発現を示した細胞のこの分野全体で、すべてのセルが同様の染色強度（ 図2B」）で見ることができることを実証した。サイトカインUPDに応答して調節されるタンパク質は、...

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

Upright Imaging of Drosophila Egg Chambers

The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

のアップライトイメージング<em&gt;ショウジョウバエ</em&gt;エッグチェンバース

Drosophila melanogaster oogenesis provides an ideal context for studying varied developmental processes since the ovary is relatively simple in ...

upright-imaging-of-drosophila-egg-chambers

Research

JoVE Journal

Developmental Biology

Upright Imaging of Drosophila Egg Chambers

7.6K ビュー.  University of Maryland Baltimore County. The upright imaging method described in this protocol allows for the detailed visualization of the poles of a developing Drosophila melanogaster egg. This end-on view provides a new perspective into the arrangements and morphologies of multiple cell types in the follicular epithelium.

ビデオ: Upright Imaging of Drosophila Egg Chambers

Live-imaging of the Drosophila Pupal Eye

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track during live cell imaging. These processes include: retinal rotation, cell growth and organismal movement. Additionally, irregularities in the topology of the epithelium, including subtle bumps and folds often introduced as the pupa is prepared for imaging, make it challenging to acquire in-focus images of more than a few ommatidia in a single focal plane. The workflow outlined here remedies these issues, allowing easy analysis of cellular processes during Drosophila pupal eye development. Appropriately-staged pupae are arranged in an imaging rig that can be easily assembled in most laboratories. Ubiquitin-DE-Cadherin:GFP and GMR-GAL4&#8211;driven UAS-&#945;-catenin:GFP are used to visualize cell boundaries in the eye epithelium 1-3. After deconvolution is applied to fluorescent images captured at multiple focal planes, maximum projection images are generated for each time point and enhanced using image editing software. Alignment algorithms are used to quickly stabilize superfluous motion, making individual cell behavior easier to track.

Live-imaging of the Drosophila Pupal Eye

This protocol presents an efficient method for imaging the live Drosophila pupal eye neuroepithelium. This method compensates for tissue movement and uneven topology, enhances visualization of cell boundaries through the use of multiple GFP-tagged junction proteins, and uses an easily-assembled imaging rig.

のライブイメージング<em&gt;ショウジョウバエ</em&gt;蛹アイ

Inherent processes of Drosophila pupal development can shift and distort the eye epithelium in ways that make individual cell behavior difficult to track ...

発生生物学

Imaging Calcium in Drosophila at Egg Activation

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. One aspect of egg activation, conserved across all organisms examined, is a change in the intracellular concentration of calcium (Ca2+) often termed a 'Ca2+ wave'. While the speed and number of oscillations of the Ca2+ wave varies between species, the change in intracellular Ca2+ is key in bringing about essential events for embryonic development. These changes include resumption of the cell cycle, mRNA regulation, cortical granule exocytosis, and rearrangement of the cytoskeleton.
In the mature Drosophila egg, activation occurs in the female oviduct prior to fertilization, initiating a series of Ca2+-dependent events. Here we present a protocol for imaging the Ca2+ wave in Drosophila. This approach provides a manipulable model system to interrogate the mechanism of the Ca2+ wave and the downstream changes associated with it.

Imaging Calcium in Drosophila at Egg Activation

This article describes an adaptable ex vivo protocol for visualizing Ca2+ during egg activation in Drosophila.

内イメージングカルシウム<em&gt;ショウジョウバエ</em&gt;卵活性化で

Egg activation is a universal process that includes a series of events to allow the fertilized egg to complete meiosis and initiate embryonic development. ...

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 ...

生物学

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A remarkable example is given by sensory epithelia, where cells of the same or distinct identities are brought together via cell-cell adhesion showing highly organized planar patterns. Cells align to one another in the same direction and display equivalent polarity over large distances. This organization of the mature epithelia is established over the course of morphogenesis. To understand how the planar arrangement of the mature epithelia is achieved, it is crucial to track cell orientation and growth dynamics with high spatiotemporal fidelity during development in vivo. Robust analytical tools are also essential to identify and characterize local-to-global transitions. The Drosophila pupa is an ideal system to evaluate oriented cell shape changes underlying epithelial morphogenesis. The pupal developing epithelium constitutes the external surface of the immobile body, allowing long-term imaging of intact animals. The protocol described here is designed to image and analyze cell behaviors at both global and local levels in the pupal abdominal epidermis as it grows. The methodology described can be easily adapted to the imaging of cell behaviors at other developmental stages, tissues, subcellular structures, or model organisms.

Imaging and Analysis of Tissue Orientation and Growth Dynamics in the Developing Drosophila Epithelia During Pupal Stages

This protocol is designed for the imaging and analysis of the dynamics of cell orientation and tissue growth in the Drosophila abdominal epithelia as the fruit fly undergoes metamorphosis. The methodology described here can be applied to the study of different developmental stages, tissues, and subcellular structures in Drosophila or other model organisms.

プパル期のショウジョウバエ上皮の組織オリエンテーションと成長ダイナミクスのイメージングと解析

Within multicellular organisms, mature tissues and organs display high degrees of order in the spatial arrangements of their constituent cells. A ...

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a well-characterized model organism, has benefited from the use of light and electron microscopy to understand gene function at the level of cells and tissues. The application of imaging platforms that allow for an understanding of gene function at the level of the entire intact organism would further enhance our knowledge of genetic mechanisms. Here a whole animal imaging method is presented that outlines the steps needed to visualize Drosophila at any developmental stage using microcomputed tomography (&#181;-CT). The advantages of &#181;-CT include commercially available instrumentation and minimal hands-on time to produce accurate 3D information at micron-level resolution without the need for tissue dissection or clearing methods. Paired with software that accelerate image analysis and 3D rendering, detailed morphometric analysis of any tissue or organ system can be performed to better understand mechanisms of development, physiology, and anatomy for both descriptive and hypothesis testing studies. By utilizing an imaging workflow that incorporates the use of electron microscopy, light microscopy, and &#181;-CT, a thorough evaluation of gene function can be performed, thus furthering the usefulness of this powerful model organism.

Whole Animal Imaging of Drosophila melanogaster using Microcomputed Tomography

A protocol is presented that allows for the visualization of intact Drosophila melanogaster at any stage of development using microcomputed tomography.

微小断層撮影による ショウジョウバエメラノガスター の動物全体イメージング

Biomedical imaging tools permit investigation of molecular mechanisms across spatial scales, from genes to organisms. Drosophila melanogaster, a ...

Visualizing the Effects of Oxidative Damage on Drosophila Egg Chambers using Live Imaging

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ribonucleoprotein particle movement, mRNA localization, organelle movement, and cytoskeletal dynamics. There are several methods for live imaging that have been developed. Due to the fact that each method involves dissecting individual ovarioles placed in media or halocarbon oil, cellular damage due to hypoxia and/or physical manipulation will inevitably occur over time. One downstream effect of hypoxia is to increase oxidative damage in the cells. The purpose of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries after induction of controlled cellular damage. Here, we use hydrogen peroxide to induce cellular oxidative damage and give examples of the effects of such damage on two subcellular structures, mitochondria and Clu bliss particles. However, this method is applicable to any subcellular structure. The limitations are that hydrogen peroxide can only be added to aqueous media and would not work for imaging that uses halocarbon oil. The advantages are that hydrogen peroxide is readily available and inexpensive, acts quickly, its concentrations can be modulated, and oxidative damage is a good approximation of damage caused by hypoxia as well as general tissue damage due to manipulation.

The objective of this protocol is to use live imaging to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in Drosophila ovaries.

ライブイメージングを用いたショウジョウバエ卵チャンバに対する酸化損傷の効果の可視化

Live imaging of Drosophila melanogaster ovaries has been instrumental in understanding a variety of basic cellular processes during development, including ...

Nuclear Migration in the Drosophila Oocyte

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton rearrangements, or polarity patterning within the cells. When studying oocyte nucleus positioning, live-imaging techniques are essential to capture the dynamic events of this process. The Drosophila egg chamber is a multicellular structure and an excellent model system to study this phenomenon because of its large size and availability of numerous genetic tools. During Drosophila mid-oogenesis, the nucleus migrates from a central position within the oocyte to adopt an asymmetric position mediated by microtubule-generated forces. This migration and positioning of the nucleus are necessary to determine the polarity axes of the embryo and the subsequent adult fly. One characteristic of this migration is that it occurs in three dimensions (3D), creating a necessity for live imaging. Thus, to study the mechanisms that regulate nuclear migration, we have developed a protocol to culture the dissected egg chambers and perform live imaging for 12 h by time-lapse acquisitions using spinning-disk confocal microscopy. Overall, our conditions allow us to preserve Drosophila egg chambers alive for a long period of time, thereby enabling the completion of nuclear migration to be visualized in a large number of samples in 3D.

Nuclear Migration in the Drosophila Oocyte

In Drosophila, the oocyte nucleus undergoes microtubule-dependent migration during oogenesis. Here, we describe a protocol that was developed to follow the migration by performing live imaging on egg chambers ex-vivo. Our procedure maintains egg chambers alive for 12 h to acquire multi-position 3D time-lapse movies using spinning-disk confocal microscopy.

ショ ウジョウバエ・ ウーサイトにおける核移動

Live cell imaging is particularly necessary to understand the cellular and molecular mechanisms that regulate organelle movements, cytoskeleton ...

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 ...

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death processes that are generally assumed to be intrinsically irreversible. Promoting anastasis could in principle rescue or preserve injured cells that are difficult to replace such as cardiomyocytes or neurons, thereby facilitating tissue recovery. Conversely, suppressing anastasis in cancer cells, undergoing apoptosis after anti-cancer therapies, may ensure cancer cell death and reduce the chances of recurrence. However, these studies have been hampered by the lack of tools for tracking the fate of cells that undergo anastasis in live animals. The challenge is to identify the cells that have reversed the cell death process despite their morphologically normal appearance after recovery. To overcome this difficulty, we have developed Drosophila and mammalian CaspaseTracker biosensor systems that can identify and permanently track the anastatic cells in vitro or in vivo. Here, we present in vivo protocols for the generation and use of the CaspaseTracker dual biosensor system to detect and track anastasis in Drosophila melanogaster after transient exposure to cell death stimuli. While conventional biosensors and protocols can label cells actively undergoing apoptotic cell death, the CaspaseTracker biosensor can permanently label cells that have recovered after caspase activation - a hallmark of late-stage apoptosis, and simultaneously identify active apoptotic processes. This biosensor can also track the recovery of the cells that attempted other forms of cell death that directly or indirectly involved caspase activity. Therefore, this protocol enables us to continuously track the fate of these cells and their progeny, facilitating future studies of the biological functions, molecular mechanisms, physiological and pathological consequences, and therapeutic implications of anastasis. We also discuss the appropriate controls to distinguish cells that undergo anastasis from those that display non-apoptotic caspase activity in vivo.

Detecting Anastasis In Vivo by CaspaseTracker Biosensor

Anastasis is technically challenging to detect in vivo because the cells that have reversed the cell death process can be morphologically indistinguishable from normal healthy cells. Here we describe protocols for detecting and tracking cells that undergo anastasis in live animals by using our newly developed in vivo CaspaseTracker biosensor system.

アナスタシスはIn Vivo CaspaseTracker バイオ センサーによる検出

Anastasis (Greek for &#8220;rising to life&#8221;) is a recently discovered cell recovery phenomenon whereby dying cells can reverse late-stage cell death ...

医学

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.

のアップライトイメージング

要約

さらに動画を探す

この動画の章

のライブイメージング

内イメージングカルシウム

のためのメゾスコピック蛍光トモグラフィー生体内イメージングショウジョウバエ

プパル期のショウジョウバエ上皮の組織オリエンテーションと成長ダイナミクスのイメージングと解析

微小断層撮影によるショウジョウバエメラノガスターの動物全体イメージング

ライブイメージングを用いたショウジョウバエ卵チャンバに対する酸化損傷の効果の可視化

ショウジョウバエ・ウーサイトにおける核移動

の直立イメージングショウジョウバエ胚

アナスタシスはIn Vivo CaspaseTracker バイオセンサーによる検出

ショウジョウバエの蛹の腹部の免疫組織化学

のアップライトイメージング

要約

さらに動画を探す

この動画の章

のライブイメージング

内イメージングカルシウム

のためのメゾスコピック蛍光トモグラフィー生体内イメージングショウジョウバエ

プパル期のショウジョウバエ上皮の組織オリエンテーションと成長ダイナミクスのイメージングと解析

微小断層撮影による ショウジョウバエメラノガスター の動物全体イメージング

ライブイメージングを用いたショウジョウバエ卵チャンバに対する酸化損傷の効果の可視化

ショ ウジョウバエ・ ウーサイトにおける核移動

の直立イメージングショウジョウバエ胚

アナスタシスはIn Vivo CaspaseTracker バイオ センサーによる検出

ショウジョウバエの蛹の腹部の免疫組織化学

微小断層撮影によるショウジョウバエメラノガスターの動物全体イメージング

ショウジョウバエ・ウーサイトにおける核移動

アナスタシスはIn Vivo CaspaseTracker バイオセンサーによる検出