We appreciate and would like to thank the Howard Hughes Medical Institute for the HHMI Professor award to U.B. which made this project possible. We thank the college at the University of California, Los Angeles for providing facilities and teaching infrastructure support for this work. The work was also supported with funding from Midwestern University and a generous donation from the Charity Fidelity Gift Fund. We thank John VandenBrooks for comments on the manuscript and Krista Pearman for her technical assistance.

この手順では、2日間の処置である。 1日目（2時間+解剖時間） 1.蛹アイディスク解剖<ol><li>解剖用の蛹を選択します。 注：解剖する蛹の年齢は実験的なニーズによって決定されます。しかし、細胞の形態を検査した場合、これはしばしば、ビデオに示す蛹の年齢である25℃、蛹殻形成（APF）の後42時間で行われる。濡れた絵筆で（0時間APF考えられる）白い蛹を収集し、25℃に維持し、新しいフライ食品バイアル中で（収集時間に基づいて）時系列順に並べ。できるだけ近い蛹間の時間差を維持するために、この同じ時系列順に熟成蛹を解剖。 </li><li>シリコーン解剖板上のリン酸緩衝生理食塩水（PBS、pH7.4で、レシピの材料を参照のこと）の低下に蛹を転送する。 </li><li>鉗子やピアスと蛹のケースの上部を持ち、Lを開く鋭いピンセットで蛹ケースのower部分。新たに形成された穴にケースから蛹を削除します。 </li><li>蛹でハサミで斜めカット、半ば胸部を作る。破片を除去またはPBSの新しいドロップに蛹ヘッドを移す。ピペット送風管を使用してクリーニングし（詳細は材料および図1を参照）、必要に応じて追加のPBSを加える。脱水症状を避けるために確認してください。 </li><li>しっかりとグリップピンセットで蛹頭部キューティクルの最上部。送風管の先端を使用して、穏やかに蛹のケースから、眼のディスクを含む、脳および他の組織を押し出すために蛹のケースに押し込み。キューティクル内の任意の組織をつかん避けるようにしてください。 </li><li> PBSを充填した送風管を使用して、穏やかに明確に分離し、脳および目のディスクを識別するために、ヘッドケースから脂肪体組織および他の破片を吹く。 注：蛹ヘッドケースに付着した脳を維持することは、研究者が彼を使用できるようになります目のディスクや脳に損傷を与えることなく、組織の将来の操作のための「ハンドル」として広告のケース。しかし、ヘッドケースから脳および目のディスクを除去することが一般的であり、これは全体的な手順および結果に有害ではない。それは頻繁に成功蛹ヘッドケース内および彼らが周囲の組織から明確に分離されるように、目からディスクと脳を抽出するために吹いてプッシュする複数の試みを取る。よりクリーンで別々の1は、目のディスクに固定から余分な組織を防ぐことができます周囲の組織からのアイディスクを得ることができます。 </li><li>切開の後、すぐに修正溶液0.5ml（材料を参照）、氷上で3ウェルガラス皿に組織を移す。数分間定着液（それは目のディスクの次のセットを分析するのにかかる時間は、通常）の表面上の組織フロート。 注記 ：これは解剖のこのラウンドで解剖された最初のアイディスクでない場合、completelyは修正液中に前のアイディスクを水没。特殊な染色手順（ 例えば 、活性酸素種、リソソームなど）を実行する場合、これは固定前に行う必要があります。これを行うために、適切な染色プロトコルを継続する前に、氷冷PBS中で解剖目ディスクを格納する。 </li><li>解剖されているすべてのアイディスクの解剖や水没に続いて、30分間室温でロッカーや岩に3ウェルガラス皿を移動します。 </li></ol> 2.免疫染色<ol><li>慎重に修正液を除去し、0.3％トリトンX-100を含むPBSを0.5mlを加える（0.3％PBT、材料を参照）、10分間、ロッカー上で組織を洗浄する。 4回の洗浄（各10分）の合計のために、この手順を3回繰り返します。 注意：ウェルから溶液を注意深く除去は、組織の損傷または損失を防止するために、解剖顕微鏡下で行うことができる。溶液はピペットで慎重に吸引することができる。我々は、ファインtippeを好むdは、開口部は、組織を吸引する可能性を低減するために平坦化されてきた使い捨てトランスファーピペット。 </li><li>第4回の洗浄の最後に、洗浄液を除去し、ウェルに（材料を参照）0.5ミリリットルブロック溶液を追加し、30分間インキュベートする。 注：この時間枠は、あなたのニーズに応じて拡張することができます。 </li><li> 30分間のブロック時間の終了時に、マイクロウェルプレートのウェルに適切な数にする（ブロック溶液中に希釈された）一次抗体溶液の10μlをピペット。 NOTE：マイクロウェルプレートの一つのウェルは蛹ケースに取り付けアイディスクの約4対または添付脳眼ディスクの8孤立対を収容する。 （：500希釈1）および抗カット（1：200希釈）代表的なデータについては、このプロトコルで使用される一次抗体は、抗ホスホチロシンです。 </li><li> 3ウェルガラス皿から一次抗体溶液を充填したマイクロウェルプレートのウェルに解剖した組織を移す。 </li><li>場所湿らせたペーパータオルの上マイクロウェルプレートは、4℃で一晩、空のピペットチップボックス（または他の類似の容器）とストア内（脱水を防ぐため）。 </li></ol> 2日目（4時間+実装時間） <ol start="6"><li> 0.3％PBT（≈0.5ml）で新しい3ウェルガラス皿にマイクロウェルプレートからの組織を移し、ロッカー上で10分間組織を洗う。 4回の洗浄（各10分）の合計のために、このステップを3回以上繰り返します</li><li>最後の洗浄液を取り出し、ウェルにブロック溶液0.5mlを加える。 30分間ブロックする。 注：この時間枠は、あなたのニーズに応じて拡張することができます。 </li><li>ブロック溶液を除去し、ウェルに二次抗体溶液（ブロック溶液中に希釈し、あなたの一次抗体および蛍光波長に適した蛍光標識された抗体から作られる）の0.5ミリリットルを追加し、カバーまたはアルミニウムで光から3ウェルガラス皿を保護箔、室温で少なくとも2時間インキュベート温度。 注：すべてのその後の工程は、同様に、露光から保護されるべきである。このインキュベーションは、一次抗体のインキュベーションと同様のセットアップで、4℃で一晩延長することができるが、抗体の保存が望まれる場合、それはまた、実験用フィルム、またはマイクロウェルプレートで覆われた3ウェルガラス皿中で行うことができる。 </li><li>抗体インキュベーションの終わりに、二次抗体溶液を除去し、ウェルに0.3％PBT 0.5mlを追加し、10分間ロッカー上洗う。 4回の洗浄（各10分）の合計に対して、この手順を繰り返します。 注：（：1,000希釈1）DAPI染色が必要な場合は、最初の洗浄を開始する前に、最初の洗浄のために使用されるPBTの500μlに（資料を参照）、DAPIストック溶液0.5μlを添加する。 </li></ol> 3.取付<ol><li>最後の洗浄の終わりには、brのを除去するための媒体としての70％グリセロールまたはPBSを使用して、スライド上の触角/眼、ディスクをマウントアインスライドの一方の側に、プール内の他の望ましくない組織。 </li><li>慎重に鉗子間の静水圧を用いて組織を動かし、または非常に優しく鉗子先端で、スライドの中心に向かって列内の目のディスクをラインアップ。 </li><li>組織の周りからすべての余分なグリセロールまたはPBSを取り外します（研究室のウィッキング作用を利用してワイプまたは鉗子は、このために働く）。 </li><li>目のディスク列の一辺に沿って封入剤を少量（≈10μl）を置き、目のディスクに封入剤を広がるように組織上カバースリップを置く。 </li><li>スライドが完全に広がる組織/封入を可能にするために1〜2分間放置します。 </li><li>カバースリップのエッジの周りから余分な封入剤を拭き取り、明確なマニキュアでそれを封印。 </li><li>共焦点顕微鏡を用いて眼ディスクの蛍光画像をキャプチャします。 </li></ol>

<ol>
	<li>Ready, D. F., Hanson, T. E., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+the+Drosophila+retina,+a+neurocrystalline+lattice.">Development of the Drosophila retina, a neurocrystalline lattice.</a> Dev Biol. 53 (2), 217-240 (1976).</li><li>Wolff, T., Ready, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+beginning+of+pattern+formation+in+the+Drosophila+compound+eye:+the+morphogenetic+furrow+and+the+second+mitotic+wave.">The beginning of pattern formation in the Drosophila compound eye: the morphogenetic furrow and the second mitotic wave.</a> Development. 113 (3), 841-850 (1991).</li><li>Carthew, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+the+Drosophila+eye.">Pattern formation in the Drosophila eye.</a> Curr Opin Genet Dev. 17 (4), 309-313 (2007).</li><li>Bao, S., Cagan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preferential+adhesion+mediated+by+Hibris+and+Roughest+regulates+morphogenesis+and+patterning+in+the+Drosophila+eye.">Preferential adhesion mediated by Hibris and Roughest regulates morphogenesis and patterning in the Drosophila eye.</a> Dev cell. 8 (6), 925-935 (2005).</li><li>Grzeschik, N. A., Knust, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IrreC/rst-mediated+cell+sorting+during+Drosophila+pupal+eye+development+depends+on+proper+localisation+of+DE-cadherin.">IrreC/rst-mediated cell sorting during Drosophila pupal eye development depends on proper localisation of DE-cadherin.</a> Development. 132 (9), 2035-2045 (2005).</li><li>Sprecher, S. G., Desplan, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switch+of+rhodopsin+expression+in+terminally+differentiated+Drosophila+sensory+neurons.">Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons.</a> Nature. 454 (7203), 533-537 (2008).</li><li>Zuker, C. S., Cowman, A. F., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+structure+of+a+rhodopsin+gene+from+D.+melanogaster.">Isolation and structure of a rhodopsin gene from D. melanogaster.</a> Cell. 40 (4), 851-858 (1985).</li><li>Voas, M. G., Rebay, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signal+integration+during+development:+insights+from+the+Drosophila+eye.">Signal integration during development: insights from the Drosophila eye.</a> Dev Dyn. 229 (1), 162-175 (2004).</li><li>Hsiao, H. Y., 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=Dissection+and+immunohistochemistry+of+larval,+pupal+and+adult+Drosophila+retinas.">Dissection and immunohistochemistry of larval, pupal and adult Drosophila retinas.</a> J Vis Exp. , 4347 (2012).</li><li>Call, G. B., 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=Genomewide+clonal+analysis+of+lethal+mutations+in+the+Drosophila+melanogaster+eye:+comparison+of+the+X+chromosome+and+autosomes.">Genomewide clonal analysis of lethal mutations in the Drosophila melanogaster eye: comparison of the X chromosome and autosomes.</a> Genetics. 177 (2), 689-697 (2007).</li><li>Chen, J., 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=Discovery-based+science+education:+functional+genomic+dissection+in+Drosophila+by+undergraduate+researchers.">Discovery-based science education: functional genomic dissection in Drosophila by undergraduate researchers.</a> PLoS Biol. 3 (2), 59 (2005).</li><li>Wolff, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+techniques+for+pupal+and+larval+Drosophila+eyes.">Dissection techniques for pupal and larval Drosophila eyes.</a> CSH Protoc. 2007, (2007).</li><li>Williamson, W. R., Hiesinger, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+developing+and+adult+Drosophila+brains+and+retinae+for+live+imaging.">Preparation of developing and adult Drosophila brains and retinae for live imaging.</a> J Vis Exp. 37, 1936 (2010).</li><li>Xu, T., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+genetic+mosaics+in+developing+and+adult+Drosophila+tissues.">Analysis of genetic mosaics in developing and adult Drosophila tissues.</a> Development. 117 (4), 1223-1237 (1993).</li><li>Duffy, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4+system+in+Drosophila:+a+fly+geneticist's+Swiss+army+knife.">GAL4 system in Drosophila: a fly geneticist's Swiss army knife.</a> Genesis. 34 (1-2), 1-15 (2002).</li></ol>

The authors have nothing to disclose.

While it appears that the process is simple and easy to perform, in reality, this technique requires a great deal of practice to master. Routinely, we start students off by learning to dissect and mount third instar eye discs12, which are much easier to work with. This practice helps to develop an appropriate dissection position of the arms, hands and fingers13 so that manipulation of the forceps under the dissecting microscope is stable, easy and experienced. In essence, the practice period shou...

キイロショウジョウバエ ：発達と細胞生物学の分野が大幅にモデル生物の影響を受けてきた。このモデルの中では、目のディスクの研究はシグナル伝達、細胞生物学やその他の分野に関する知識を大いに貢献してきました。後半に第三の幼虫齢目のディスクが広く研究し、それが発達期間の一連のスナップショットを与えるように、利用するための強力なモデルであるされている、独自のシグナリング分子、およびプロセスとのそれぞれが、形態形成溝は、目のディスクを横切って進むにつれて8。しかし、更なる発展の蛹の段階に発達過程の理解を拡大する必要性がある。蛹のアイディスク3-7の研究がなされてきた一方で、私たちの知識は3齢目のディスクに行われている仕事の幅広さに近づかない。これは、蛹のアイディスク解剖で大きな困難に一部起因している。したがって、プレゼンテーション解剖の適切な方法が大幅にこの分野の研究を展開することができます。 簡単に、特に中旬蛹期間を中心に、解剖された蛹のアイディスク開発中の段階がありますが、他の期間は、はるかに困難な解剖べきである。このプロトコルは、普遍的にすべての蛹の発育時間枠のために使用することができる蛹目ディスクを切開するための1つの方法を表している。このプロトコルは、midpupal時点から目ディスクを分析するための簡単かつ迅速に方法を示す別のプロトコル9の代替として使用することができる。このプロトコルは、もともと撮影さと機能ゲノミクス（URCFG）蛹眼の解剖の手法で10,11でUCLAの学部研究コンソーシアムで高度な学部学生を訓練するために開発されました。多くの学部学生がこのような厳しいテクニックを学ぶためにこのビデオと方法を利用することができました。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Phosphate-buffered saline (PBS, pH 7.4)</td>
			<td></td>
			<td></td>
			<td>80g NaCl, 2g&#160; KCl, 14.4g Na2HPO4, 2.4g KH2PO4, Bring volume to 1 l, adjust the pH to 7.4, autoclave or filter sterilize, dilute to 1X PBS with autoclaved ddH2O before using.</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Promega</td>
			<td>H5142</td>
			<td>Caution: Irritant! Wear gloves.</td>
		</tr>
		<tr>
			<td>0.3% PBT</td>
			<td></td>
			<td></td>
			<td>1.5 ml of Triton X-100, 500 ml 1X PBS.</td>
		</tr>
		<tr>
			<td>37% formaldehyde solution</td>
			<td>Fisher Scientific</td>
			<td>F75P1GAL</td>
			<td>Caution: Toxic, probable human carcinogen! Wear gloves.&#160;</td>
		</tr>
		<tr>
			<td>Fix Solution</td>
			<td></td>
			<td></td>
			<td>(&#8776;4% Formaldehyde in PBS) 50 &#956;l of 37% Formaldehyde, 450 &#956;l 1XPBS, make fresh before use</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Rockland antibodies &#38; assays</td>
			<td>B304</td>
			<td>Aliquot in 1 ml volumes and store at -80C</td>
		</tr>
		<tr>
			<td>Block Solution</td>
			<td></td>
			<td></td>
			<td>10% NGS in PBT.&#160; This can be made and stored at 4 &#176;C for a few days prior to use.</td>
		</tr>
		<tr>
			<td>DAPI stock solution</td>
			<td>Life Technologies</td>
			<td>D3571</td>
			<td>For coutnerstaining nuclei. Prepare a 1 mg/ml solution with ddH2O.</td>
		</tr>
		<tr>
			<td>VectaShield Mounting Medium</td>
			<td>Vector Labs</td>
			<td>H-1000</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma</td>
			<td>G5516</td>
			<td>For mounting. Prepare 70% dilution with ddH2O.</td>
		</tr>
		<tr>
			<td colspan="4">Equipment</td>
		</tr>
		<tr>
			<td>Nutating mixer</td>
			<td>VWR</td>
			<td>82007-202</td>
			<td>Used to rock tissue in 3 well glass dish</td>
		</tr>
		<tr>
			<td>SylGard 182 Silicone Elastomer Kit</td>
			<td>Krayden</td>
			<td>NC9897184</td>
			<td>Used to make silicone dissection dish</td>
		</tr>
		<tr>
			<td>Silicone dissecting dish</td>
			<td></td>
			<td></td>
			<td>Mix Sylgard elastomer kit (above) according to directions gently (to avoid bubbles). Pour mixture into Petri dish (any size). Allow SylGard to cure overnight in 37 &#176;C incubator.</td>
		</tr>
		<tr>
			<td>3 well glass dish</td>
			<td>Corning</td>
			<td>7220-85</td>
			<td>The 3 well variety of these are no longer available, this is the 9 well product.</td>
		</tr>
		<tr>
			<td>72 well microwell minitray</td>
			<td>Nunc</td>
			<td>438733</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp forceps (Dumont #55)</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td></td>
		</tr>
		<tr>
			<td><a name="RANGE!A19">Vannas-type Micro Scissors, Straight, 5mm blade</a></td>
			<td>Ted Pella</td>
			<td>1346</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm Borosilicate glass capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100-4</td>
			<td>Pull with needle puller to make fine point tip that allows a small stream of PBS to flow.</td>
		</tr>
		<tr>
			<td>Disposable Transfer Pipets, Fine Tip</td>
			<td>Samco Scientific</td>
			<td>231</td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Tubing dimensions given are inner diameter (ID) x outer diameter (OD) x wall thickness in inches</td>
		</tr>
		<tr>
			<td>PVC tubing (1/8 x 3/16 x 1/32)</td>
			<td>Nalgene</td>
			<td>8000-0010</td>
			<td rowspan="3">Use these with pulled needle to assemble the blower tube as shown in Figure 2.</td>
		</tr>
		<tr>
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			<td>Saint Gobain Performance Plastics</td>
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</table>

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.

このプロトコルの使用例として、異なる抗体で免疫染色midpupalを示す結果（42時間APF 25°Cで）眼のディスクはホスホチロシン残基に対する抗体を使用することにより、図2に示されている、細胞の膜とすることができる観測された（ 図2A）。これはmidpupal段階の前に発生する最後のパターニングプロセス以下蛹眼の個眼細胞の規則的な配列を同定するために用いることができる。別?...

Watch this Scientific Journal Video about Dissection and Mounting of Drosophila Pupal Eye Discs at JoVE.com

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 ...

dissection-and-mounting-of-drosophila-pupal-eye-discs

Research

JoVE Journal

Biology

Dissection and Mounting of Drosophila Pupal Eye Discs

12.1K ビュー.  University of California at Los Angeles (UCLA). 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.

ビデオ: Dissection and Mounting of Drosophila Pupal Eye Discs

Dissection of Drosophila Ovaries

ショウジョウバエの卵巣の解剖

生物学

Dissection of Imaginal Discs from 3rd Instar Drosophila Larvae

This protocol demonstrates the dissection technique used for isolating imaginal discs from drosophila larvae at the 3rd instar stage. Methods for fixing the tissue after saying and removing the wing, leg, and eye discs are demonstrated directly on a microscope slide for subsequent visualization.

3齢幼虫ショウジョウバエの幼虫から成虫ディスクの解剖

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

ショウジョウバエ幼虫NMJの解剖

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

の解剖と染色ショウジョウバエ幼虫卵巣

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Drosophila Pupal Abdomen Immunohistochemistry

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

Drosophila Pupal Abdomen Immunohistochemistry

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

ショウジョウバエの蛹の腹部の免疫組織化学

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

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

の蛹ケースを画像化<em&gt;キイロショウジョウバエ</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

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 ...

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes differentiation from a stem cell population, mitotic amplification, and meiosis. In addition, post-meiotic germ cells undergo a dramatic morphological reshaping process as well as a global epigenetic reconfiguration of the germ line chromatin&#8212;the histone-to-protamine switch.
Studying the role of a protein in post-meiotic spermatogenesis using mutagenesis or other genetic tools is often impeded by essential embryonic, pre-meiotic, or meiotic functions of the protein under investigation. The post-meiotic phenotype of a mutant of such a protein could be obscured through an earlier developmental block, or the interpretation of the phenotype could be complicated. The model organism Drosophila melanogaster offers a bypass to this problem: intact testes and even cysts of germ cells dissected from early pupae are able to develop ex vivo in culture medium. Making use of such cultures allows microscopic imaging of living germ cells in testes and of germ-line cysts. Importantly, the cultivated testes and germ cells also become accessible to pharmacological inhibitors, thereby permitting manipulation of enzymatic functions during spermatogenesis, including post-meiotic stages.
The protocol presented describes how to dissect and cultivate pupal testes and germ-line cysts. Information on the development of pupal testes and culture conditions are provided alongside microscope imaging data of live testes and germ-line cysts in culture. We also describe a pharmacological assay to study post-meiotic spermatogenesis, exemplified by an assay targeting the histone-to-protamine switch using the histone acetyltransferase inhibitor anacardic acid. In principle, this cultivation method could be adapted to address many other research questions in pre- and post-meiotic spermatogenesis.

Ex vivo Culture of Drosophila Pupal Testis and Single Male Germ-line Cysts: Dissection, Imaging, and Pharmacological Treatment

This protocol describes the dissection and cultivation of intact testes and germ-line cysts from Drosophila melanogaster pupae. This method allows microscopic observation of spermatogenesis ex vivo. Furthermore, we describe a pharmacological assay of the effect of inhibitors on specific stages of germ-cell development in pupal testes.

解剖、イメージング、および薬理学的治療： ショウジョウバエ蛹精巣や独身男性生殖ライン嚢胞のex vivo培養

During spermatogenesis in mammals and in Drosophila melanogaster, male germ cells develop in a series of essential developmental processes. This includes ...

Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

解剖やイトヨ鰓スケルトンのフラットマウント

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary changes. In particular, the adult stage is a rich source of morphological traits for interspecific comparison, including wing pigmentation comparison. To study developmental differences among species, detailed observation and appropriate staging are required for precise comparison. Here we describe protocols for staging of pupal periods and quantification of wing pigmentation in a polka-dotted fruit fly, Drosophila guttifera. First, we describe the method for detailed morphological observation and definition of pupal stages based on morphologies. This method includes a technique for removing the puparium, which is the outer chitinous case of the pupa, to enable detailed observation of pupal morphologies. Second, we describe the method for measuring the duration of defined pupal stages. Finally, we describe the method for quantification of wing pigmentation based on image analysis using digital images and ImageJ software. With these methods, we can establish a solid basis for comparing developmental processes of adult traits during pupal stages.

Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera

Protocols for staging pupal periods and measurement of wing pigmentation of Drosophila guttifera are described. Staging and quantification of pigmentation provide a solid basis for studying developmental mechanisms of adult traits and enable interspecific comparison of trait development.

ステージング蛹期間およびショウジョウバエ guttiferaの翼色素沈着測定のためのメソッド

Diversified species of Drosophila (fruit fly) provide opportunities to study mechanisms of development and genetic changes responsible for evolutionary ...