We thank the Bloomington Stock Center (Bloomington, USA), Dirk Bohmann, Katja Br&#252;ckner and Istvan Ando for fly stocks, and antibodies. We thank Marek Jindra and Colin Donohoe for comments on the manuscript. This work was supported by the Sofja Kovalevskaja Award to M.U. from the Alexander von Humboldt Foundation and DFG project UH243/1-1 to M.U. from the German Research Foundation.

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The authors declare that they have no conflict of interest.

ショウジョウバエにおける遺伝的モザイクを生成するための技術は、遺伝子機能33を解析し、操作するための最も洗練されたツールの一つです。それはアイレスエンハンサーは9,18アクティブになっている組織では、空間的に制限された方法で視覚的にマークされ、遺伝学的に定義されたクローン、 すなわちの誘導を可能にするようeyFLP-MARCMシステムは、?...

がんは発症率と死亡率の劇的全世界、特に高齢者の間で、増加している疾患の最も遺伝的に不均質なグループの1つを表します。がんは、固有の腫瘍抑制メカニズムを脱出し、制御不能に分割し、腫瘍開始細胞からクローン由来します。死と分化を抑制しつつ、協働の成長、増殖および運動性を促進する遺伝子病変の段階的な蓄積は非常に、悪性の転移性と致命的な腫瘍に初期良性の異常増殖を変換します。これは、遺伝的変化に加えて、腫瘍の進行は、その微小環境における腫瘍と複数の細胞型（ 例えば 、線維芽細胞、免疫細胞および内皮細胞）の間、周囲の間質とのクロストークの変化を必要とすることが明らかになりました。腫瘍 - 間質相互作用を含む悪性形質転換の根底にある分子の原則を理解することはプリベンを開発するために非常に重要です化と早期スクリーニング戦略だけでなく、癌の転移と薬剤耐性に対抗するための新しい効果的な治療法。 フルーツフライキイロショウジョウバエは、その高速な世代時間に起因する癌研究1-4のための魅力的なシステム、ハエと人間の間のノードのシグナリング顕著保全、のほぼすべての遺伝子の操作を容易に高度な遺伝子ツールの限られた遺伝的冗長性と豊富になってきています一時的および空間的に制限された方法。変化する悪性腫瘍の遺伝的に定義された腫瘍が再現MARCM技術5を用いて、そうでなければ、野生型組織における前駆細胞のサブセットにおいてgain-及び機能喪失変異を導入することにより、ショウジョウバエで操作することができます。 MARCMツールは、FLP / FRT（FLPリコンビナーゼ/ FLP認識ターゲット）はFLPアウト7およびGAL4 / UAS（上流活性化配列）8標的遺伝子との有糸分裂組換え6を媒介組み合わせ発現系9。 dsRNAを誘導される遺伝子サイレンシングのための癌遺伝子または蛍光タンパク質のcDNAまたは反転DNA反復を含む任意のUASベースの導入遺伝子のこのメソッド式で、特定の遺伝子座と組み換えによるGal4のリプレッサーを失った細胞のクローンに制限されます（ 図1A）。クローンパッチは緑色蛍光（GFP）または赤色蛍光タンパク質でマークされた（ 例えば、RFP、DsRedを、mCherryを）を容易に開発し、単離し、分析を通じて追跡することができます。重要なことには、それらの動作は、直接隣接し、野生型組織と比較することができます。このように、遺伝的病変の細胞自律的かつ非自律的効果に関連する質問が便利に研究することができます。哺乳動物に類似し、唯一の複数の発癌性病変が組み合わされたクローンは、 ショウジョウバエにおいて、悪性になると、哺乳動物の癌の主要な特徴を再現します。彼らは、アポトーシスを回避炎症を誘発し、不滅とINVAになり、過剰に増殖しますSIVEは、最終的にホスト10-17を殺します。 ここでは、MARCM技術を用いたショウジョウバエの幼虫の目/触角と脳組織に遺伝学的に定義されたクローン性腫瘍を生成するためのプロトコルについて説明します。この方法は、 アイレスエンハンサー（eyFLP）18,19の制御下で酵母FLPリコンビナーゼを発現するMARCMテスター株に依存しています。このように、GFP標識クローンはperipodial柱状EADの上皮および胚および幼生期（ 図1A、Bおよび参照20）を通じて、脳の神経上皮の両方で生成されます。神経上皮が差別視葉ニューロンを産生する神経芽細胞を生じさせるながらEADは、大人の目、アンテナと頭部カプセルに発展するにつれクローンは、簡単に成人期まで続くことができます。 モザイク組織の大規模な、分子の機能的および表現型の特徴付けを容易にするために、我々デスクライブ3齢幼虫からEADと脳の解剖のためのプロトコルは、3つの異なるアプリケーションのためにそれらを処理する方法を示しています。（ⅰ）トランスジェニック蛍光レポーターおよび免疫染色の検出、腫瘍侵襲性との（iii）の分析の（ii）の定量化遺伝子発現は、定量的リアルタイムPCR（QRT-PCR）またはハイスループットのmRNA配列（mRNAの-SEQ）（ 図1C）を用いて変更します。 免疫プロトコルは、特定の抗体と目的の任意のタンパク質を可視化するために使用することができます。トランスジェニック蛍光の転写レポーターは、特定のシグナル伝達経路の活性に便利で正確な時空間情報を提供します。細胞系統特異的レポーターは、他の一方で、モザイク組織内および個別の遺伝子型の腫瘍のうち、細胞集団における定性的および定量的変化を反映します。侵襲的な行動の定量化は、遺伝子型間の腫瘍の悪性度の比較が容易秒。最後に、RNA単離のためにモザイクEADの収集と処理を説明するプロトコルは、それぞれ定量RT-PCRおよびゲノムワイドのmRNA-seqの、続いて逆転写などの小型の両方大規模なダウンストリームアプリケーションに適しています。これらのアッセイから得られた定性的および定量的なデータは、クローン性腫瘍の社会的行動への新たな洞察を提供します。さらに、それらは個々の遺伝子、遺伝子ネットワークと異なる段階における腫瘍微小環境と腫瘍形成の側面の役割に関する機能研究のための強固な基盤を作り出します。

<table><tbody><tr><td>Agar</td><td>Gew&amp;uuml;rzm&amp;uuml;hle Brecht, Eggenstein, Germany</td><td>00262-0500</td><td>Fly food recipe: Prepare 20 L fly food with 160 g agar, 360 g yeast, 200 g soy flour, 1.6 kg yellow cornmeal, 1.2 L malt extract, 300 ml light corn syrup, 130 ml propionic acid and 200 ml 15% nipagin. Fly food should be cooked for 1 hour at 85&amp;nbsp;&amp;deg;C.</td></tr><tr><td>Corn syrup</td><td>Grafschafter Krautfabrik Josef Schmitz KG, Meckenheim, Germany</td><td>01939</td><td></td></tr><tr><td>Propionic acid</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>6026.1</td><td></td></tr><tr><td>Cornmeal</td><td>ReformKontor GmbH, Zarrentin, Germany</td><td>4010155063948</td><td></td></tr><tr><td>Malt extract</td><td>CSM Deutschland GmbH, Bremen, Germany</td><td>728985</td><td></td></tr><tr><td>Soy flour</td><td>Stockmeier Food GmbH, Herford, Germany</td><td>1000246441010</td><td></td></tr><tr><td>Yeast</td><td>Werner Ramspeck GmbH, Schwabach, Germany</td><td>210099K</td><td></td></tr><tr><td>Methyl-4-benzoate/ Nipagin</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>H5501</td><td>Prepare a 15% stock solution with 70% EtOH</td></tr><tr><td>Drosophila fly food vials</td><td>Kisker Biotech, Steinfurt, Germany</td><td>789008</td><td></td></tr><tr><td>Vial plugs</td><td>K-TK e.K., Retzstadt, Germany</td><td>1002</td><td></td></tr><tr><td>Drosophila fly food bottles</td><td>Greiner Bio-One, Frickenhausen, Germany</td><td>960177</td><td></td></tr><tr><td>Bottle plugs</td><td>K-TK e.K., Retzstadt, Germany</td><td>1002 S</td><td></td></tr><tr><td>Poly(vinyl alcohol) 4-88/[-CH&lt;sub&gt;2&lt;/sub&gt;CHOH-]&lt;sub&gt;n&lt;/sub&gt;</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>81381</td><td>Mounting medium recipe: Dissolve 6 g Poly(vinyl alcohol) 4-88 in 39&amp;nbsp;ml Millipore H&lt;sub&gt;2&lt;/sub&gt;O, 6 ml 1 M Tris (pH 8.5) and 12.5 ml glycerol. Stir overnight at 50&amp;nbsp;&amp;deg;C and centrifuge 20&amp;nbsp;min at 5,000 rpm. Add DABCO to the supernatant to get a final concentration of 2.5%. Store aliquots at -20&amp;nbsp;&amp;deg;C.</td></tr><tr><td>1,4-Diazabicyclo[2.2.2]octane/ DABCO&amp;nbsp;</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>D2522</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>9002-93-1</td><td></td></tr><tr><td>Phosphate-buffered saline/ PBS</td><td></td><td></td><td>137 mM NaCl, 2.7 mM KCl, 10 mM Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt; and 1.8 mM KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, pH 7.4 in Millipore H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>PBST</td><td></td><td></td><td>0.1% Triton X-100 in PBS</td></tr><tr><td>Paraformaldehyde/ PFA</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>158127</td><td>4% PFA fixative recipe: Dissolve 4&amp;nbsp;g PFA in 80 ml Millipore H&lt;sub&gt;2&lt;/sub&gt;O on a magnetic stirrer plate heated to 55&amp;nbsp;&amp;deg;C. Add 1 M NaOH dropwise until all PFA particles are dissolved. Add 10 ml 10x PBS and adjust the pH to 7.4 with 1 M HCl. Mix in 100 &amp;micro;l Triton X-100 and fill up with Millipore H&lt;sub&gt;2&lt;/sub&gt;O to 100 ml. Store aliquots at -20 &amp;deg;C. Avoid repeated thawing. (CAUTION: PFA is highly toxic. Prepare the PFA fixative in a fume hood. Wear a self-contained breathing apparatus, gloves and clothing. Avoid contact with skin, eyes or mucous membranes.)</td></tr><tr><td>Bovine Serum Albumin/ BSA</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>A3059</td><td>Blocking solution recipe: Dissolve 0.3% BSA in PBST</td></tr><tr><td>Fluorescently labeled phalloidin</td><td>Invitrogen, Karlsruhe, Germany, Molecular Probes</td><td>A12379, A22283, A22284</td><td>Phalloidin stock solution: Dissolve powder (300 U) in 1.5 ml Methanol. Store at -20 &amp;deg;C. For staining, use in dilution 1:250 (A12379, A22283) and 1:100 (A22284) in PBST.</td></tr><tr><td>4&amp;rsquo;,6-Diamidino-2-phenylindol Dihydrochlorid/ DAPI</td><td>Carl Roth GmbH, Karlsruhe, Germany</td><td>6335</td><td>DAPI staining solution: Prepare a stock solution of 5 mg/ml in Millipore H&lt;sub&gt;2&lt;/sub&gt;O and store aliquots at 4 &amp;deg;C. Used in dilution 1:1,000 in PBST.</td></tr><tr><td>Mouse anti-H2 antibody</td><td>Kurucz et al., 2003</td><td></td><td>Used in dilution 1:500 in blocking solution</td></tr><tr><td>Cy5 AffiniPure Donkey Anti-Mouse IgG (H+L)</td><td>Jackson ImmunoResearch, Suffolk, UK</td><td>715-175-151</td><td>Used in dilution 1:500 in blocking solution</td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools, Heidelberg, Germany</td><td>11295-10</td><td></td></tr><tr><td>Glass embryo dish (30&amp;nbsp;mm)</td><td>Thermo Scientific, Schwerte, Germany</td><td>E90</td><td></td></tr><tr><td>Tungsten needles</td><td>Fine Science Tools, Heidelberg, Germany</td><td>10130-20</td><td></td></tr><tr><td>Nickel Plated Pin Holder</td><td>Fine Science Tools, Heidelberg, Germany</td><td>26018-17</td><td></td></tr><tr><td>Microscope slides</td><td>VWR, Darmstadt, Germany</td><td>631-1553</td><td></td></tr><tr><td>Coverslips 22 x 22 mm (#1 Menzel-Gl&amp;auml;ser)</td><td>VWR, Darmstadt, Germany</td><td>631-1336</td><td></td></tr><tr><td>Coverslips 24 x 50 mm (0.13-0.16&amp;nbsp;mm)</td><td>Thermo Scientific, Schwerte, Germany</td><td>1076371</td><td></td></tr><tr><td>Kimtech Science Precision Wipes</td><td>Thermo Scientific, Schwerte, Germany</td><td>06-677-70</td><td></td></tr><tr><td>Dissecting stereomicroscope</td><td>Olympus, Hamburg, Germany</td><td>SZX7 (DF PLAPO 1X-4 Japan)</td><td></td></tr><tr><td>Fluorescent stereomicroscope</td><td>Olympus, Hamburg, Germany</td><td>SZX16 (SDF PLAPO 0.8X Japan) with DP72 CCD camera</td><td>Equipped with the narrow blue bandpass filter set for excitation of GFP other blue excitable fluorochromes (excitation filter BP460-480 nm, barrier filter BA495-540 nm) and narrow green excitation and longpass barrier filter for RFP and other green excitable fluorochromes (excitation filter BP530-550, barrier filter BA575IF). Software: Olympus cellSens Standard 1.11.</td></tr><tr><td>Confocal microscope</td><td>Olympus, Hamburg, Germany</td><td>FV1000</td><td>Equipped with inverted IX81 microscope. Objectives: 20&amp;times; UPlan S-Apo (NA 0.85), 40&amp;times; UPlanFL (NA 1.30) and 60&amp;times; UPlanApo (NA 1.35). Lasers: UV laser Diode LD405 (50&amp;nbsp;mW), Argon laser, multi-line 457/(476)/ 488/515 (40&amp;nbsp;mW), Yellow/Green laser diode LD560 (15&amp;nbsp;mW) and Red Laser diode 635 (20&amp;nbsp;mW). Software: Fluoview 2.1c Software</td></tr><tr><td>Drosophila cooled incubator</td><td>Ewald Innovationstechnik GmbH, Bad Nenndorf, Germany</td><td>&amp;nbsp;Sanyo MIR553</td><td></td></tr><tr><td>Squirt bottle</td><td>VWR, Darmstadt, Germany</td><td>215-8105</td><td></td></tr><tr><td>BD Clay Adams Nutator Mixer</td><td>VWR, Darmstadt, Germany</td><td>15172-203</td><td></td></tr><tr><td>ELMI Digital Rocking Shaker</td><td>VWR, Darmstadt, Germany</td><td>DRS-12</td><td></td></tr><tr><td>5 PRIME Isol-RNA Lysis Reagent</td><td>VWR, Darmstadt, Germany</td><td>2302700</td><td>The protocol is compatible with other TRIzol-based reagents, &lt;em&gt;e.g.&lt;/em&gt;, TRIreagent from Sigma (Cat. Nr.T9424), TRIzol reagent from Thermofisher (Cat. Nr. 15596).</td></tr><tr><td>Diethyl pyrocarbonate/ DEPC</td><td>Sigma-Aldrich, Deisenhofen, Germany</td><td>D5758</td><td>Dilute DEPC 1:1,000 in Millipore H&lt;sub&gt;2&lt;/sub&gt;O, stir overnight and autoclave.</td></tr><tr><td>UltraPure Phenol:Chloroform:Isoamyl Alcohol (25:24:1)</td><td>ThermoFischer Scientific, Invitrogen, Karlsruhe, Germany</td><td>15593-031</td><td></td></tr><tr><td>Chloroform</td><td>Merck, Darmstadt, Germany</td><td>102445</td><td></td></tr><tr><td>TURBO DNase (2 U/&amp;micro;l)</td><td>Thermo Scientific, Schwerte, Germany</td><td>AM2238</td><td></td></tr><tr><td>Invitrogen UltraPure Glycogen</td><td>ThermoFischer Scientific, Invitrogen, Karlsruhe, Germany</td><td>10-814-010</td><td></td></tr><tr><td>Sodium acetate trihydrate</td><td>VWR, Darmstadt, Germany</td><td>27652.232</td><td>Prepare a 3 M Sodium acetate solution with DEPC-H&lt;sub&gt;2&lt;/sub&gt;O and adjust the pH to 5.2</td></tr><tr><td>2-Propanol</td><td>Merck, Darmstadt, Germany</td><td>109634</td><td></td></tr><tr><td>Ethanol</td><td>Merck, Darmstadt, Germany</td><td>100983</td><td>Prepare a 75% EtOH dilution with DEPC-H&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>UV/Vis-Spectrophotometre NanoDrop ND-8000</td><td>Thermo Scientific, Schwerte, Germany</td><td>ND-8000</td><td></td></tr><tr><td>Eppendorf Microcentrifuge (Refrigerated)</td><td>Thermo Scientific, Schwerte, Germany</td><td>5417R</td><td></td></tr><tr><td>Experion RNA StdSens Analysis Kit</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>7007103</td><td></td></tr><tr><td>Experion Automated Electrophoresis Station</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>7007010</td><td></td></tr><tr><td>SuperScript III Reverse Transcriptase</td><td>Thermo Scientific, Schwerte, Germany</td><td>18080044</td><td></td></tr><tr><td>Oligo d(T) Primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>Prepare 100 &amp;micro;M stock solutions in DEPC-H&lt;sub&gt;2&lt;/sub&gt;O and store at -20 &amp;deg;C</td></tr><tr><td>dNTP Mixture</td><td>Takara Bio Europe/Clonetech, Saint-Germain-en-Laye, France</td><td>4030</td><td>Store aliquots of 25 &amp;micro;l at -20&amp;deg;C</td></tr><tr><td>CFX96 Touch Real-Time PCR Detection System</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>1855195</td><td></td></tr><tr><td>iQ SYBR Green Supermix</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>170-8882</td><td></td></tr><tr><td>Hard-Shell PCR Plates 96-well, thin wall</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>HSP9601</td><td></td></tr><tr><td>Microseal &#039;B&#039; Film</td><td>Bio-Rad Laboratories GmbH, M&amp;uuml;nchen, Germany</td><td>MSB1001</td><td></td></tr><tr><td>&lt;em&gt;rp49&lt;/em&gt; Forward primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>5&#039; TCC TAC CAG CTT CAA GAT GAC 3&#039;</td></tr><tr><td>&lt;em&gt;rp49&lt;/em&gt; Reverse primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>5&#039; CAC GTT GTG CAC CAG GAA CT 3&#039;</td></tr><tr><td>&lt;em&gt;mmp1&lt;/em&gt; Forward primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>5&#039; AGG GCG ACA AGT ACT ACA AGC TGA 3&#039;</td></tr><tr><td>&lt;em&gt;mmp1&lt;/em&gt; Reverse primer</td><td>Integrated DNA Technologies, Leuven, Belgium</td><td></td><td>5&#039; ACG TCT TGC CGT TCT TGT AGG TGA 3&#039;</td></tr></tbody></table>

the drosophila imaginal disc tumor model: visualization and quantification of gene expression and tumor invasiveness using genetic mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

クローンの3種類が誘導されたショウジョウバエ EADで定義された遺伝子型のGFP-マークされたパッチを生成するために、eyFLP-MARCM技術の可能性を実証するために：（1）コントロールのみGFPを発現する、（2）悪性腫瘍が小さいの発癌性型を発現します腫瘍抑制遺伝子の落書きのホモ接合損失（ のScrib 1）の背景には、Gタンパク質のRas（ラス<sup...

Watch this Scientific Journal Video about The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics at JoVE.com

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

このプロトコルは、 ショウジョウバエの眼/触角成虫（EAD）に蛍光マークされ、遺伝的に定義されたクローンの腫瘍を生成する方法を示します。なお、第3齢幼虫とどのように遺伝子発現の変化と腫瘍侵襲性を可視化し、定量化するためにそれらを処理するからEADと脳を分析する方法について説明します。

ザ<em&gt;ショウジョウバエ</em&gt;成虫盤腫瘍モデル：遺伝的モザイクを用いた遺伝子発現と腫瘍侵襲性の可視化と定量化

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

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JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

11.0K ビュー.  University of Cologne. このプロトコルは、 ショウジョウバエの眼/触角成虫（EAD）に蛍光マークされ、遺伝的に定義されたクローンの腫瘍を生成する方法を示します。なお、第3齢幼虫とどのように遺伝子発現の変化と腫瘍侵襲性を可視化し、定量化するためにそれらを処理するからEADと脳を分析する方法について説明します。 

ビデオ: The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Mosaic Zebrafish Transgenesis for Functional Genomic Analysis of Candidate Cooperative Genes in Tumor Pathogenesis

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers. To robustly and efficiently identify oncogenic &#8220;drivers&#8221; among these tumors and define their complex relationships with concurrent genetic alterations during tumor pathogenesis remains a daunting task. Recently, zebrafish have emerged as an important animal model for studying human diseases, largely because of their ease of maintenance, high fecundity, obvious advantages for in vivo imaging, high conservation of oncogenes and their molecular pathways, susceptibility to tumorigenesis and, most importantly, the availability of transgenic techniques suitable for use in the fish. Transgenic zebrafish models of cancer have been widely used to dissect oncogenic pathways in diverse tumor types. However, developing a stable transgenic fish model is both tedious and time-consuming, and it is even more difficult and more time-consuming to dissect the cooperation of multiple genes in disease pathogenesis using this approach, which requires the generation of multiple transgenic lines with overexpression of the individual genes of interest followed by complicated breeding of these stable transgenic lines. Hence, use of a mosaic transient transgenic approach in zebrafish offers unique advantages for functional genomic analysis in vivo. Briefly, candidate transgenes can be coinjected into one-cell-stage wild-type or transgenic zebrafish embryos and allowed to integrate together into each somatic cell in a mosaic pattern that leads to mixed genotypes in the same primarily injected animal. This permits one to investigate in a faster and less expensive manner whether and how the candidate genes can collaborate with each other to drive tumorigenesis. By transient overexpression of activated ALK in the transgenic fish overexpressing MYCN, we demonstrate here the cooperation of these two oncogenes in the pathogenesis of a pediatric cancer, neuroblastoma that has resisted most forms of contemporary treatment.

The goal of this study is to demonstrate how the mosaic transgenesis strategy can be used in zebrafish to rapidly and efficiently assess the relative contributions of multiple oncogenes in tumor initiation and progression in vivo.

腫瘍の病因に候補協同遺伝子の機能ゲノム解析のためのモザイクゼブラフィッシュ遺伝子導入

Comprehensive genomic analysis has uncovered surprisingly large numbers of genetic alterations in various types of cancers.  To robustly and efficiently ...

発生生物学

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue organization. Drosophila melanogaster has emerged as a popular experimental model system in cancer biology to study the genetic and cellular mechanisms of tumorigenesis. In particular, genetic tools for Drosophila imaginal discs (developing epithelia in larvae) enable the creation of transformed pro-tumor cells within a normal epithelial tissue, a situation similar to the initial stages of human cancer. A recent study of tumorigenesis in Drosophila wing imaginal discs, however, showed that tumor initiation depends on the tissue-intrinsic cytoarchitecture and the local microenvironment, suggesting that it is important to consider the region-specific susceptibility to tumorigenic stimuli in evaluating tumor phenotypes in imaginal discs. To facilitate phenotypic analysis of tumor progression in imaginal discs, here we describe a protocol for genetic experiments using the GAL4-UAS system to induce neoplastic tumors in wing imaginal discs. We further introduce a diagnosis method to classify the phenotypes of clonal lesions induced in imaginal epithelia, as a clear classification method to discriminate various stages of tumor progression (such as hyperplasia, dysplasia, or neoplasia) had not been described before. These methods might be broadly applicable to the clonal analysis of tumor phenotypes in various organs in Drosophila.

Induction and Diagnosis of Tumors in Drosophila Imaginal Disc Epithelia

Mosaic clone analysis in Drosophila imaginal disc epithelia is a powerful model system to study the genetic and cellular mechanisms of tumorigenesis. Here we describe a protocol to induce tumors in Drosophila wing imaginal discs using the GAL4-UAS system, and introduce a diagnosis method to classify the tumor phenotypes.

腫瘍の誘導および診断<em&gt;ショウジョウバエ</em&gt; Imaginal Disc Epithelia

In the early stages of cancer, transformed mutant cells show cytological abnormalities, begin uncontrolled overgrowth, and progressively disrupt tissue ...

がん研究

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 expression is rarely tissue-specific enough to allow for precise correlation of the circuit with behavioral readouts. To overcome this major hurdle, we recently developed the FINGR method to achieve a more restrictive Gal4 expression in the tissue of interest. The FINGR method has three components: 1) the traditional Gal4/UAS system; 2) a set of FLP/FRT-mediated Gal80 converting tools; and 3) enhancer-trap FLP (ET-FLP). Gal4 is used to define the primary neural circuitry of interest. Paring the Gal4 with a UAS-effector, such as UAS-MJD78Q or UAS-Shits, regulates the neuronal activity, which is in turn manifested by alterations in the fly behavior. With an additional UAS-reporter such as UAS-GFP, the neural circuit involved in the specific behavior can be simultaneously mapped for morphological analysis. For Gal4 lines with broad expression, Gal4 expression can be restricted by using two complementary Gal80-converting tools: tubP&gt;Gal80&gt; ('flip out') and tubP&gt;stop&gt;Gal80 ('flip in'). Finally, investigators can turn Gal80 on or off, respectively, with the help of tissue-specific ET-FLP. In the flip-in mode, Gal80 will repress Gal4 expression wherever Gal4 and ET-FLP intersect. In the flip-out mode, Gal80 will relieve Gal4 repression in cells in which Gal4 and FLP overlap. Both approaches enable the restriction of the number of cells in the Gal4-defined circuitry, but in an inverse pattern. The FINGR method is compatible with the vast collection of Gal4 lines in the fly community and highly versatile for traditional clonal analysis and for neural circuit mapping. In this protocol, we demonstrate the mapping of FLP expression patterns in select ET-FLPx2 lines and the effectiveness of the FINGR method in photoreceptor cells. The principle of the FINGR method should also be applicable to other genetic model organisms in which Gal4/UAS, Gal80, and FLP/FRT are used.

Mapping and Application of Enhancer-trap Flippase Expression in Larval and Adult Drosophila CNS

We describe a Flippase-induced intersectional Gal80/Gal4 repression (FINGR) method, allowing tissue-specific FLP to determine Gal80 expression patterns. Wherever Gal4 and FLP overlap, Gal4 expression is turned on (Gal80 flipped out) or off (Gal80 flipped in). The FINGR method is versatile for clonal analysis and neural circuit mapping.

エンハンサートラップフリッパーゼの幼虫における発現と成人のマッピングとその応用ショウジョウバエ CNS

The Gal4/ UAS binary method is powerful for gene and neural circuitry manipulation in Drosophila. For most neurobiological studies, however, Gal4 ...

神経科学

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

の形態素解析ショウジョウバエ</em遺伝モザイクを使用>幼虫末梢感覚ニューロンの樹状突起と軸索

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Drosophila has illuminated our understanding of the genetic basis of normal development and disease for the past several decades and today it continues to contribute immensely to our understanding of complex diseases 1-7. Progression of tumors from a benign to a metastatic state is a complex event 8 and has been modeled in Drosophila to help us better understand the genetic basis of this disease 9. Here I present a simple protocol to genetically induce, observe and then analyze the progression of tumors in Drosophila larvae. The tumor induction technique is based on the MARCM system 10 and exploits the cooperation between an activated oncogene, RasV12 and loss of cell polarity genes (scribbled, discs large and lethal giant larvae) to generate invasive tumors 9. I demonstrate how these tumors can be visualized in the intact larvae and then how these can be dissected out for further analysis. The simplified protocol presented here should make it possible for this technique to be utilized by investigators interested in understanding the role of a gene in tumor invasion.

A Protocol for Genetic Induction and Visualization of Benign and Invasive Tumors in Cephalic Complexes of Drosophila melanogaster

Cooperation between an activated oncogene like RASV12 and mutations in cell polarity genes like scribbled, result in tumor growth in Drosophila where tumor cells also display invasive behaviors. Here a simple protocol for the induction and observation of the benign and invasive tumors is presented.

遺伝的誘導との頭部の複合体における良性および浸潤性腫瘍の可視化のためのプロトコル<em&gt;キイロショウジョウバエ</em

Drosophila has illuminated our  understanding of the genetic basis of normal development and disease for the  past several decades and today it continues ...

医学

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 ...

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 ...

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, compromising downstream analyses. Considering the complex and dynamic cellular associations existing within many tissues, in order to recapitulate the in vivo interactions thorough molecular analysis one must be able to analyze specific cell populations within their native context. Laser-mediated microdissection can achieve this goal, allowing unambiguous identification and successful harvest of cells of interest under direct microscopic visualization while maintaining molecular integrity. We have applied this technology to analyse gene expression within defined areas of the developing Drosophila wing disc, which represents an advantageous model system to study growth control, cell differentiation and organogenesis. Larval imaginal discs are precociously subdivided into anterior and posterior, dorsal and ventral compartments by lineage restriction boundaries. Making use of the inducible GAL4-UAS binary expression system, each of these compartments can be specifically labelled in transgenic flies expressing an UAS-GFP transgene under the control of the appropriate GAL4-driver construct. In the transgenic discs, gene expression profiling of discrete subsets of cells can precisely be determined after laser-mediated microdissection, using the fluorescent GFP signal to guide laser cut. 
Among the variety of downstream applications, we focused on RNA transcript profiling after localised RNA interference (RNAi). With the advent of RNAi technology, GFP labelling can be coupled with localised knockdown of a given gene, allowing to determinate the transcriptional response of a discrete cell population to the specific gene silencing. To validate this approach, we dissected equivalent areas of the disc from the posterior (labelled by GFP expression), and the anterior (unlabelled) compartment upon regional silencing in the P compartment of an otherwise ubiquitously expressed gene. RNA was extracted from microdissected silenced and unsilenced areas and comparative gene expression profiling determined by quantitative real-time RT-PCR. We show that this method can effectively be applied for accurate transcriptomics of subsets of cells within the Drosophila imaginal discs. Indeed, while massive disc preparation as source of RNA generally assumes cell homogeneity, it is well known that transcriptional expression can vary greatly within these structures in consequence of positional information. Using localized fluorescent GFP signal to guide laser cut, more accurate transcriptional analyses can be performed and profitably applied to disparate applications, including transcript profiling of distinct cell lineages within their native context.

Laser Microdissection Applied to Gene Expression Profiling of Subset of Cells from the Drosophila Wing Disc

Laser microdissection was applied to analyse gene expression profiling in specific compartments of Drosophila wing disc subjected to localised RNAi in vivo. RNA extracted from equivalent areas of silenced and unsilenced compartments was analysed by quantitative RT-PCR to determine comparative gene expression profiling within the context of native tissue microecology.

から細胞のサブセットの遺伝子発現プロファイリングへの応用レーザーマイクロダイセクションショウジョウバエウィングディスク

Heterogeneous nature of tissues has proven to be a limiting factor in the amount of information that can be generated from biological samples, ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

解剖や成虫の免疫染色から<em&gt;キイロショウジョウバエ</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

解剖と実装<em&gt;ショウジョウバエ</em&gt;蛹アイディスク

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

ザ

要約

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腫瘍の病因に候補協同遺伝子の機能ゲノム解析のためのモザイクゼブラフィッシュ遺伝子導入

腫瘍の誘導および診断

エンハンサートラップフリッパーゼの幼虫における発現と成人のマッピングとその応用ショウジョウバエ CNS

の形態素解析ショウジョウバエ幼虫末梢感覚ニューロンの樹状突起と軸索

遺伝的誘導との頭部の複合体における良性および浸潤性腫瘍の可視化のためのプロトコル

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

から細胞のサブセットの遺伝子発現プロファイリングへの応用レーザーマイクロダイセクションショウジョウバエウィングディスク

解剖や成虫の免疫染色から

解剖と実装