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Biologia

収穫と準備ショウジョウバエ胚

Published: May 20, 2009
doi:
10.3791/1347
, ,
1Department of Biological Sciences,University of Illinois, 2Department of Biological Sciences,Vanderbilt University

Summary

この手法は、免疫組織化学または電気生理学的記録のためのショウジョウバエ胚の神経筋肉組織を公開します。それは神経筋発達の初期段階でイベントを勉強したり、孵化することができない変異体では電気生理学を実行する際に役立つ。

Abstract

ショウジョウバエ胚発生と機能的神経科学の両方の研究のための最高の遺伝的モデルです。伝統的に、これらのフィールドは非常に大部分が独立の歴史と科学的なコミュニティと、互いに分離されています。しかし、これらは通常異なる学問分野間のインターフェイスは、神経回路形成の最終段階、機能化学シナプスの機能的電気信号特性と分化の買収の基礎となる発生プログラムです。このインタフェースは、調査のために非常に重要な領域です。 ショウジョウバエでは、機能開発のこれらのフェーズは、胚発生の最後の3分の1の間（25℃）<8時間の期間中に発生します。この後期発達期間が長くタフな、不浸透性の表皮クチクラの堆積により調査に難治性と考えられた。画期的な進歩は、局所的に後期胚の制御解剖を有効にするためにキューティクルに適用できる水重合外科接着剤のアプリケーションでした。背側縦切開で、胚は実験的な調査に腹神経索と体壁の筋肉組織を露出させる、フラットなレイアウトすることができます。このシステムは、大きく胚性シナプス形成を損なうため、シナプスの接続と機能的なシナプス伝達特性の仕様と差別化を支配する分子メカニズムを明らかに遺伝的変異体を分離し、特徴付けるために使用されています。

Protocol

パート1：設備＆備品良い解剖顕微鏡は、胚の解剖が必要です。40X倍率は25倍アイピース最大限に倍率を増加させると、示唆されている。 ファイン鉗子（番号5）は胚と胚のdevitellinizationの手動選択のために必要とされています。 微細なガラス針を作成したり変更したりする機器が必要です。プルガラス針は解剖のために必要とされています。我々は解剖（長い最後）のた?…

Discussion

胚の正確なステージングは​​、原因だけで数時間の時間の経過とともに機能的特性の急激な成熟に重要です。いくつかの問題は、このステージングを複雑にする。最初に、ほとんどの研究者は、タイミング卵の段階の胚に産むを使用していますが、産卵時には、別の条件（Broadieら1992）で、動物から動物に非常に異なることがあります。特に、限られた食事療法で女性は前の敷設に長時間?…

Acknowledgements

KBはNIH助成金GM54544によってサポートされています。

Materials

Material Name Tipo Company Catalogue Number Comment
Embryo Collection Cages   Genesee Scientific (www.flystuff.com) 59-100 (for 60 mm dish; other sizes available) Cages can also be home made using punctured tri-pour beakers, as shown in video
Sylgard 184   Dow Corning Available from various companies Surgical glue adheres better to sylgard-coated coverslips
Fine glass tubing, outer diameter 1.0-1.5 mm   various   For pulling into fine glass needles for dissection and tubes for glue delivery
Plastic tubing, to attach to glass pipette for mouth suction and glue application   Tygon   Tubing inner diameter needs to match glass outer diameter.
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Tags

DrosophilaEmbryosElectrophysiological RecordingProceduresGenetic ModelEmbryonic DevelopmentFunctional NeuroscienceDevelopmental ProgramsElectrical Signaling PropertiesChemical SynapsesNeural Circuit FormationInvestigationLate Developmental PeriodEpidermal CuticleSurgical GlueControlled DissectionDorsal Longitudinal IncisionVentral Nerve CordBody Wall MusculatureGenetic MutantsSynapse FormationMolecular Mechanisms
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Featherstone, D. E., Chen, K., Broadie, K. Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures. J. Vis. Exp. (27), e1347, doi:10.3791/1347 (2009).
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