Name: methods for staging pupal periods and measurement of wing pigmentation of drosophila guttifera
Uploaded: 2018-01-24T15:00:00
Description: Watch this Scientific Journal Video about Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera at JoVE.com

直行用ヒューズ、秉ソクジン撮影、メンタリングの阿形とエリザベス中島英語を編集するための彼の援助のためのはえの在庫を提供する、ショーン b. キャロルとヴェルナー ・ トーマスに感謝します。この仕事は、費によって支えられた 17 K 19427 と武田科学振興財団。

1. フライ ストック
<ol><li>次のプロトコルのすべてのショウジョウバエ guttiferaを使用します。</li>
	<li>プラスチック バイアルを使用 (直径 25 mm × 高さ 96 mm) とセルロース プラグ (直径 23 mm × 高さ 26 mm) 在庫メンテナンスのため。標準的なコーンミール、砂糖、酵母、寒天食品を使用し、従って文書説明この種2の他の 3 つの代替レシピ。 注: D. guttifera (在庫数 ...

<ol>
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ここで述べたい蛹の段階の定義のプロトコル削除の詳細な観察は、囲蛹、蛹の期間とd. guttiferaの翼に黒い斑点の強度の測定を測定します。これらのプロトコルは、多くのショウジョウバエのため適用することができ、フライ種、特に翼色素沈着との関連。
詳細な観察と発達するより詳細なイベントの説明により、さらに段階の細分化。多くの場合、郭清を?...

ショウジョウバエの形態学的特性のいくつかは種1,2,3,4、5の中で多様化します。質問をアプローチすることができますどの形態の多様性のこれらの形態の発生のメカニズムを比較することによって発生します。このような形態の例として、幼虫の毛、大人のセックスの櫛、外部生殖器装置、腹部色素沈着翼色素沈着6,7,8,9,10,11,12,13,14,15. 大人の間で形態学的差異を研究する蛹の段階の観察と解析、ので、重要後期幼虫段階で大人の特性の運命を決定し、その後形態蛹の期間中。
ショウジョウバエ、発達生物学で「時間 APF」(蛹形成後の時間) 蛹のステージ16を示す一般的な方法です。このシステムは蛹形成後の絶対時間を採用、日常的実験のため非常に便利です。しかし、発達速度、蛹の間で異なる場合があり、エピジェネティックな遺伝子またはアイソレータケージのわずかな違いによって影響を受ける可能性があります、したがって、同じ蛹形成後の絶対時間は保証しません蛹は、同時に発達段階。多くの場合、形態学的特徴によって定義される段階は複数の個人を比較することをお勧めです。特に、正確なステージングと対応する (相同) 段階間の比較、種の比較が必要です。
ベイン ブリッジと Bownes17認識 20 蛹化 (P15(ii)) に P1 はショウジョウバエ蛹の形態学的特徴に基づきます。このステージングは、形態学的発達ステージング18の最も広く使われているシステムです。以前の研究では、翼の色素沈着研究19のための基礎を確立するショウジョウバエ guttiferaの蛹ステージングを行った。D. guttiferaは黒の水玉柄は、その翼、翼色素形成20のモデル種の 1 つです。記載されている形態学的基準に紹介がベイン ブリッジと Bownes の研究17、我々 は、シリアル観察19、ベイン ブリッジと Bownes の段階の期間の推定を使用する代わりに、ステージの期間を直接測定周波数を観察しました。ここで蛹のステージングのメソッドと福富ら19で使用されるショウジョウバエの蛹の段階の期間の測定について述べる。
翼の色素沈着の発生機序を研究、蛹または大人の段階で色素沈着が発生するときに知っている必要があります。福富ら。19は、翼画像の画像解析による蛹および大人の段階で色素の光学密度 (ODs) を定量化します。ショウジョウバエ翼の色素沈着は、黒いメラニン21の蓄積に起因すると考えられます。ODs の定量化, グレイ スケール画像や ImageJ のソフトウェア (https://imagej.nih.gov/ij/) の22が使用されました。認識してスポット特有の色素沈着 (ΔOD) を定量化、我々 はスポット内 OD からスポット外 OD を減算します。この方法をするためには、再現性と客観的ランドマークとして翼静脈を用いる OD 測定場所を決定してください。この資料でこのショウジョウバエ guttiferaの翼色素形成の定量化の方法くわしく述べる。

<table>
	<tbody>
		<tr>
			<td>Drosophila guttifera</td>
			<td>The Drosophila Species Stock Center at the U.C. San Diego</td>
			<td>15130-1971.10</td>
			<td>Drosophila guttifera, a fruit fly species used in this article</td>
		</tr>
		<tr>
			<td>Plastic vial</td>
			<td>Hightech</td>
			<td>MKC-30</td>
			<td>Plastic vial, for fly stock maintenance</td>
		</tr>
		<tr>
			<td>Buzz plugs vial and bottle closures for glass vials</td>
			<td>Fisher Scientific</td>
			<td>AS-271</td>
			<td>Cellulose plug, for fly stock maintenance</td>
		</tr>
		<tr>
			<td>White soft sugar</td>
			<td>Mitsui Sugar</td>
			<td>J-500g</td>
			<td>White soft sugar, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Corn flour</td>
			<td>Nippon Flour Mills</td>
			<td>F</td>
			<td>Corn flour, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Corn grits - C</td>
			<td>Nippon Flour Mills</td>
			<td>GC</td>
			<td>Corn grits - C, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Agar powder</td>
			<td>Matsuki Kanten Sangyo</td>
			<td>No.602</td>
			<td>Agar powder, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Dry beer yeast</td>
			<td>Asahi Food &#38; Healthcare</td>
			<td>Y2A</td>
			<td>Dry beer yeast, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Butyl p-hydroxybenzoate</td>
			<td>Nacalai Tesque</td>
			<td>06327-02</td>
			<td>Butyl p-hydroxybenzoate, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Wako</td>
			<td>057-00456</td>
			<td>Ethanol, for standard cornmeal/sugar/yeast/agar food</td>
		</tr>
		<tr>
			<td>Flat bottom microtube</td>
			<td>Ina Optica</td>
			<td>CF-0150</td>
			<td>1.5 mL microtube, for collecting pupae</td>
		</tr>
		<tr>
			<td>CAPSULEFUGE</td>
			<td>Tomy</td>
			<td>PMC-060</td>
			<td>Mini microcentrifuge, for collecting pupae</td>
		</tr>
		<tr>
			<td>Sterilized Schale NB</td>
			<td>Sansei Medical</td>
			<td>01-013</td>
			<td>Plastic Petri dish (diameter 90 mm x height 15 mm)</td>
		</tr>
		<tr>
			<td>Serum tube rack</td>
			<td>Iwaki</td>
			<td>9796-050</td>
			<td>Used as a moist chamber, for observation of pupa</td>
		</tr>
		<tr>
			<td>Corning Falcon Easy-Grip tissue culture dish</td>
			<td>Corning</td>
			<td>353001</td>
			<td>Plastic Petri dish (diameter 35 mm x height 10 mm)</td>
		</tr>
		<tr>
			<td>Falcon standard tissue culture dish</td>
			<td>Corning</td>
			<td>353002</td>
			<td>Plastic Petri dish (diameter 60 mm x height 15 mm)</td>
		</tr>
		<tr>
			<td>Push-pin</td>
			<td>Kokuyo</td>
			<td>51233709</td>
			<td>Push-pin, for making pinholes on the microtube lid</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Olympus</td>
			<td>SZX16</td>
			<td>Stereomicroscope, for morphological observation</td>
		</tr>
		<tr>
			<td>Digital camera</td>
			<td>Olympus</td>
			<td>DSE-330-A</td>
			<td>Digital camera, for imaging</td>
		</tr>
		<tr>
			<td>NICETACK double sided tape</td>
			<td>Nichiban</td>
			<td>NW-15SF</td>
			<td>Double sided tape, for removing puparium</td>
		</tr>
		<tr>
			<td>Dumont #5 forceps</td>
			<td>Fine Science Tools</td>
			<td>11252-20</td>
			<td>Forceps, for removing puparium</td>
		</tr>
		<tr>
			<td>Van Gogh VISUAL Paint brush</td>
			<td>Talens Japan</td>
			<td>GWVR-#5/0</td>
			<td>Paint brush, for removing puparium</td>
		</tr>
		<tr>
			<td>Greiner CELLSTAR 12 well cell culture plate</td>
			<td>Merck</td>
			<td>665-180</td>
			<td>12-well cell culture plate, for measuring durations of pupal periods</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Wako</td>
			<td>191-01665</td>
			<td>NaCl, for PBS</td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Nacalai Tesque</td>
			<td>285-14</td>
			<td>KCl, for PBS</td>
		</tr>
		<tr>
			<td>Na2HPO4&#183;12H2O</td>
			<td>Wako</td>
			<td>196-02835</td>
			<td>Na2HPO4&#183;12H2O, for PBS</td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Nacalai Tesque</td>
			<td>28721-55</td>
			<td>KH2PO4, for PBS</td>
		</tr>
		<tr>
			<td>Stepped Neutral Density (ND) Filter 0.04 - 3.0</td>
			<td>Edmund Optics</td>
			<td>64-384</td>
			<td>Stepped density filter, for calibration of pigmentation measurement</td>
		</tr>
		<tr>
			<td>ImageJ software</td>
			<td>NIH</td>
			<td>1.8.0-101</td>
			<td>ImageJ software, for measurement of intensity of black spots on a wing (https://imagej.nih.gov)</td>
		</tr>
		<tr>
			<td>FINE FROST glass slide</td>
			<td>Matsunami Glass Ind</td>
			<td>FF-001</td>
			<td>Glass slide, for measurement of intensity of black spots on a wing</td>
		</tr>
		<tr>
			<td>Square microscope cover glass 18 x 18</td>
			<td>Matsunami Glass Ind</td>
			<td>C018181</td>
			<td>Cover slip, for measurement of intensity of black spots on a wing</td>
		</tr>
	</tbody>
</table>

methods for staging pupal periods and measurement of wing pigmentation of drosophila guttifera

ショウジョウバエ(ミバエ) の多様な種は、開発および進化的変化の責任遺伝子の変化のメカニズムを勉強する機会を提供します。特に、大人の段階は翼色素沈着比較を含む種間の比較形態学的形質の豊富なソースです。種の発達の違いを研究する、詳細な観察と適切なステージングが正確な比較のため必要です。ここで蛹期間のステージングと水玉フルーツ フライ、ショウジョウバエの guttiferaで翼色素沈着の定量化のプロトコルについて述べる。まず、詳細な形態学的観察と形態に基づく蛹のステージの定義の方法について述べる。このメソッドには、蛹の形態の詳細な観察を可能にする、蛹の外側キチン質ケースでは蛹殻を除去するための技術が含まれています。第二に、定義された蛹の期間の測定方法について述べる。最後に、デジタル画像や ImageJ ソフトウェアを用いた画像解析に基づく翼色素の定量法について述べる。これらのメソッドは、蛹大人形質の発達プロセスを比較するための強固な基盤を確立できます。

D. guttiferaの蛹の期間は (P1 - P15(ii); (P1、P5 の - 6、P10) の段階は図 3、およびすべての 17 段階を図 4に示すとおり 3 つ代表の画像) 17 段階に分けられます。ベイン ブリッジと Bownes17は、キイロショウジョウバエの 20 段階を認識、 d. guttiferaにいくつかこれらの段階のない適用でした。2 つの...

Watch this Scientific Journal Video about Methods for Staging Pupal Periods and Measurement of Wing Pigmentation of Drosophila guttifera at JoVE.com

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

ステージングの蛹期間およびショウジョウバエ guttiferaの翼色素沈着の測定のためのプロトコルを説明します。ステージングと色素沈着の定量化アダルト形質の発達のメカニズムを研究するための強固な基盤を提供して形質の開発に関する種間比較を有効にします。

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 ...

Research

JoVE Journal

Biology

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

High-resolution Measurement of Odor-Driven Behavior in Drosophila Larvae

ショウジョウバエの幼虫の匂い駆動型の動作の高分解能測定

Olfactory responses in Drosophila larvae have been traditionally studied in Petri dishes comprising a single peripheral odor source. In this behavioral ...

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

ショウジョウバエの幼虫のNMJからシナプス電位を記録するための電気生理学的方法

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

Live Dissection of Drosophila Embryos: Streamlined Methods for Screening Mutant Collections by Antibody Staining

のライブ解剖ショウジョウバエ胚：抗体染色によってミュータントコレクションをスクリーニングするための合理化手法

Drosophila embryos between stages 14 and 17 of embryonic development can be readily dissected to generate "fillet" preparations.  In these preparations, ...

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

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

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, ...

Drosophila Pupal Abdomen Immunohistochemistry

Drosophila Pupal Abdomen Immunohistochemistry

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 ...

Measurement of Lifespan in Drosophila melanogaster

Measurement of Lifespan in Drosophila melanogaster

における寿命の測定<em&gt;キイロショウジョウバエ</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

飛行性能の正確かつ迅速な測定のための改善された方法での<em&gt;ショウジョウバエ</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

Imaging Through the Pupal Case of Drosophila melanogaster

Imaging Through the Pupal Case of Drosophila melanogaster

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 ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Measurement of Metabolic Rate in Drosophila using Respirometry

代謝率の測定に<em&gt;ショウジョウバエ</em&gt;呼吸計測を使用した

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

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

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

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 ...