この作品は、DFGからGMTへの助成金によって支えられて

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著者らは、彼らが競合する経済的利益を持っていないことを宣言します。

モデル系としてショウジョウバエの一つの主要な利点は、単一細胞のレベルでの発達と機能の解析を可能にすることである。これは、細胞の種類の多様性は非常に高く、隣接する細胞の機能と形態が全く異なることがあります神経系に関しては特に便利です。 ここでお見せする方法は、永久染色剤や蛍光顕微鏡法による直接調べに変換することができる色素を持?...

個々の細胞レベルでの神経形態の知識は神経接続とCNS機能を理解するための重要な前提条件です。このように、神経科学の黎明期から、研究者は、単一細胞標識技術（この問題の歴史的な治療のために7を参照）を開発しようとしてきた。そのようなゴルジ染色などの古典的な方法は、神経形態の優れた解像度を提供しますが、一つは染色はランダムに発生するように、指示された方法で、神経細胞の特定の種類にラベルを付けるために求める場合は適していません。微小電極から色素の細胞内またはjuxtacellular注射することで、単一細胞を染色するための方法の開発は、特定のラベリングの要件に対処した。 ショウジョウバエへの単一ニューロン染料注入のアプリケーションが原因生物とニューロンの両方のサイズが小さい、大きな課題を提示した。 ショウジョウバエの胚のそれにもかかわらず、単一のニューロンの染色</em&gt;ニューロンはコーリー·グッドマン15の研究室では、1980年代半ばに達成された。方法は極めて有用であったし、最後の20〜30年（ 例えば、2、10）以上、ショウジョウバエの神経発達のメカニズムにいくつかの重要な洞察を提供してきましたが、多くの労働者が主な理由として、その技術的な要求のために、それを敬遠してきた。 ショウジョウバエのニューロンの標識のための遺伝的技術のより近年の可用性は、単一ニューロンの色素注入の不人気に貢献してきました。膜標的化GFPコンストラクトのGAL4指向発現は、神経形態学1、20の優れた分解能を提供することができます。しかし、この方法は、一定の制限があります：複数の細胞におけるGFPのしばしば不可避式は個々のニューロンの構造を分かりにくくすることができますとGAL4ドライバーラインは、関心のある特定の神経細胞での発現を駆動するのに利用可能でないかもしれません。 MARCM（REPを持つモザイク解析ressible細胞マーカー）法8は、個々 の細胞レベルで、本質的に任意のニューロンを標識しましたが、正常になぜならGAL80タンパク質の代謝回転の遅い胚と早期幼虫に使用することはできませんを提供することができます。 遺伝標識のこれらの制限を考えて、私たちは、 ショウジョウバエの胚において、単一ニューロンの色素注入は重要な手法のままで、広範なアプリケーションに値すると信じています。この目標を推進するために、我々はここで方法の詳細な説明を提供します。そのパワーのイラストが遅くショウジョウバエの胚14の腹部neuromeresにおける介在の完全なセットの形態の我々の最近のアカウントで提供されています。個々の神経細胞種の形態学的変動と胎児の中枢神経系における神経分節組織の原則の両方を明らかにしたこの研究では、現在使用可能な他の標識法では不可能だったでしょう。

<table border="1"><tbody><tr><td> 試薬/材料の名称 </td><td> 会社 </td><td> カタログ番号 </td><td> 注釈 </td></tr><tr><td></td><td></td><td></td><td> 試薬 </td></tr><tr><td> DAB </td><td>シグマアルドリッチ</td><td> <a href="http://www.sigmaaldrich.com/catalog/product/sigma/d5905" target="_blank">D-5905</a> </td><td> 100mMのトリス-HCl、pH 7.4で2-3 mg / mlの</td></tr><tr><td> DiIを</td><td>インビトロジェン/モレキュラープローブ</td><td> <a href="http://products.invitrogen.com/ivgn/product/D282" target="_blank">D-282</a> </td><td>エタノール中で1 mg / mlの</td></tr><tr><td> <a href="http://www.merckmillipore.com/germany/chemicals/formaldehydloesung-min-37-prozent/MDA_CHEM-104002/p_Wt6b.s1L6pAAAAEWpOEfVhTl" target="_blank">ホルムアルデヒド</a> </td><td>メルクミリポア</td><td></td><td> PBS中の7,4％ </td> &lt;/ TR&gt; <tr><td>グリセロール</td><td> <a href="http://www.carlroth.com/catalogue/" target="_blank">ロート</a> </td><td> 3783 </td><td> PBS中で70パーセント</td></tr><tr><td>ヘプタングルー</td><td>バイヤスドルフ社</td><td>チェロ31-39-30 * </td><td> caを薄める。 n-ヘプタンで1から1 </td></tr><tr><td> PBS </td><td></td><td></td><td> 1X </td></tr><tr><td>トリス</td><td> <a href="http://www.carlroth.com/catalogue/" target="_blank">ロート</a> </td><td> 4855 </td><td></td></tr><tr><td> Vectashield </td><td>ベクターラボラトリーズ</td><td> <a href="http://www.vectorlabs.com/catalog.aspx?prodID=427" target="_blank">H1000</a> </td><td></td></tr><tr><td></td><td></td><td></td><td> 機器 </td></tr><tr><td>共焦点顕微鏡</td><td>ライカ</td><td> TCS SP2 </td><td>蛍光で標識化して表示し、文書化する</td></tr><tr><td> DC -mplifier </td><td>ドラガン·コーポレーション</td><td>礎石ION-100 </td><td>ラベル付けを実行する</td></tr><tr><td>カバースリップ</td><td> <a href="http://www.menzel.de/" target="_blank">メンツェル</a> </td><td> BB018018A1 </td><td> 18 x 18のmm </td></tr><tr><td>カバースリップ</td><td> <a href="http://www.menzel.de/" target="_blank">メンツェル</a> </td><td> BB024060A1 </td><td> 24×60ミリメートル</td></tr><tr><td>解剖顕微鏡</td><td>ライカ</td><td> MZ8 </td><td>胚を準備しdisectする</td></tr><tr><td>フラットキャピラリ</td><td> <a href="http://www.hilgenberg-gmbh.de/" target="_blank">Hilgenberg</a> </td><td></td><td>外径1ミリメートル; GLAS厚さ0.1ミリメートル</td></tr><tr><td>注射毛細血管</td><td>サイエンス事業</td><td> 100 GBのTF 8P </td><td></td></tr><tr><td>マニピュレーターR </td><td>ライカ</td><td></td><td>ラベル付けを実行する</td></tr><tr><td>モデルP-97 </td><td>サターインスツルメンツ</td><td></td><td>毛細血管を引っ張る</td></tr><tr><td>オブジェクトスライド</td><td> <a href="http://www.marienfeld-superior.com/" target="_blank">Marienfeldスペリオル</a> </td><td> 1000000 </td><td></td></tr><tr><td>科学的な顕微鏡</td><td>ツァイス</td><td> Axioskop 2名言</td><td>光変換後にラベル付けを表示する</td></tr><tr><td>科学的な顕微鏡</td><td>オリンポス</td><td> BX50 </td><td>ラベル付けを実行する</td></tr><tr><td>ソニーMC3255ビデオカメラ</td><td>ソニー/ AVTホーン</td><td>ソニーMC3255 </td><td>光変換後ラベリングを記録する</td></tr></tbody></table> 表1。

labeling of single cells in the central nervous system of drosophila melanogaster

この記事では、我々は個々に親油性の蛍光膜マーカーDiIのjuxtacellular注射によるキイロショウジョウバエの胚中枢神経系の神経細胞をラベル付けする方法を説明します。この方法は、非常に詳細に神経細胞形態の可視化を可能にします。それは、CNS内の任意のセルにラベルを付けることが可能です：標的ニューロンの細胞体は、DIC光学の下やGFPなどの蛍光遺伝子マーカーの発現によって可視化されています。標識後、DIIは、透過光とDIC光学系と細胞形態の可視化を可能にすることにより光電永久茶色の染色に変換することができます。あるいは、DiI標識細胞を遺伝子導入した蛍光レポータータンパク質がcolocalisedすることを可能にする、共焦点顕微鏡で直接観察することができます。技術はそれが可能な単一細胞の解像度で変異体の表現型を解析すること、遺伝子型にかかわらず、任意の動物に使用することができます。

図4は技術の典型的な結果を示して、私たちはここで説明しています。 図4Aは、きれいにphotoconvertedたDiIを埋め単一介在ニューロンの例を示しています。それは、うまくこれらの準備が提供する詳細情報の量を示しています。非標識の周囲の組織内に標識された細胞の空間的な文脈が見えるようになり、DIC光学の下で見たときに、皮質内および神経網内の繊維投影の?...

Watch this Scientific Journal Video about Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster at JoVE.com

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

我々は、中枢神経系における単一ニューロンを標識するための技術（CNS）の提示<em&gt;ショウジョウバエ</em&gt;透過光または共焦点顕微鏡法のいずれかによる神経形態の分析を可能にする胚。

の中枢神経系における単一細胞のラベリング<em&gt;キイロショウジョウバエ</em

In this  article we describe how to individually label neurons in the embryonic CNS of Drosophila melanogaster by juxtacellular injection of the ...

labeling-single-cells-central-nervous-system-drosophila

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

12.7K ビュー.  University of Mainz. 我々は、中枢神経系における単一ニューロンを標識するための技術（CNS）の提示ショウジョウバエ透過光または共焦点顕微鏡法のいずれかによる神経形態の分析を可能にする胚。

ビデオ: Labeling of Single Cells in the Central Nervous System of Drosophila melanogaster

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

キイロショウジョウバエの幼虫中枢神経系の解剖

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

生物学

Purification of Low-abundant Cells in the Drosophila Visual System

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small numbers of cells. Utilizing this technology to study development in the Drosophila visual system, which boasts a wealth of cell type-specific genetic tools, provides a powerful approach for addressing the molecular basis of development with precise cellular resolution. For such an approach to be feasible, it is crucial to have the capacity to reliably and efficiently purify cells present at low abundance within the brain. Here, we present a method that allows efficient purification of single cell clones in genetic mosaic experiments. With this protocol, we consistently achieve a high cellular yield after purification using fluorescence activated cell sorting (FACS) (~25% of all labeled cells), and successfully performed transcriptomics analyses on single cell clones generated through mosaic analysis with a repressible cell marker (MARCM). This protocol is ideal for applying transcriptomic and genomic analyses to specific cell types in the visual system, across different stages of development and in the context of different genetic manipulations.

Here, we present a cell dissociation protocol for efficiently isolating cells present at low abundance within the Drosophila visual system through fluorescence activated cell sorting (FACS).

ショウジョウバエ視覚系における低豊富な細胞の浄化

Recent improvements in the sensitivity of next generation sequencing have facilitated the application of transcriptomic and genomic analyses to small ...

遺伝学

Cell Lineage Analyses and Gene Function Studies Using Twin-spot MARCM

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.

Here, we present a protocol for a mosaic labeling technique that permits the visualization of neurons derived from a common progenitor cell in two distinct colors. This facilitates neural lineage analysis with the capability of birth-dating individual neurons and studying gene function in the same neurons of different individuals.

ツインスポットMARCMを使用して細胞系譜の解析と遺伝子機能研究

Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological ...

発生生物学

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between cell types or even within a given type? What kinds of spatial rules do they follow? The answers to such fundamental questions in vivo have been hampered so far by a lack of tools for high resolution single cell labeling. Here, a detailed protocol to target single cells with a MultiColor FlpOut (MCFO) technique is provided. This method relies on three differently tagged reporters (HA, FLAG and V5) under UAS control that are kept silent by a transcriptional terminator flanked by two FRT sites (FRT-stop-FRT). A heat shock pulse induces the expression of a heat shock-induced Flp recombinase, which randomly removes the FRT-stop-FRT cassettes in individual cells: expression occurs only in cells that also express a GAL4 driver. This leads to an array of differently colored cells of a given cell type that allows the visualization of individual cell morphologies at high resolution. As an example, the MCFO technique can be combined with specific glial GAL4 drivers to visualize the morphologies of the different glial subtypes in the adult Drosophila brain.

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Cells display different morphologies and establish a variety of interactions with their neighbors. This protocol describes how to reveal the morphology of single cells and to investigate cell-cell interaction by using the well-established Gal4/UAS expression system.

多色 FlpOut 技術の高分解能単一細胞の形態とショウジョウバエにおけるグリア細胞の細胞間相互作用の研究への応用

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between ...

行動学

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval body wall contains several cell types that are models to study cell and tissue morphogenesis. For example tracheae are used to examine tube morphogenesis1, and the dendritic arborization (DA) sensory neurons of the Drosophila larva have become a primary system for the elucidation of general and neuron-class-specific mechanisms of dendritic differentiation2-5 and degeneration6.
The shape of dendrite branches can vary significantly between neuron classes, and even among different branches of a single neuron7,8. Genetic studies in DA neurons suggest that differential cytoskeletal organization can underlie morphological differences in dendritic branch shape4,9-11. We provide a robust immunological labeling method to assay in vivo microtubule organization in DA sensory neuron dendrite arbor (Figures 1, 2, Movie 1). This protocol illustrates the dissection and immunostaining of first instar larva, a stage when active sensory neuron dendrite outgrowth and branching organization is occurring 12,13.
In addition to staining sensory neurons, this method achieves robust labeling of microtubule organization in muscles (Movies 2, 3), trachea (Figure 3, Movie 3), and other body wall tissues. It is valuable for investigators wishing to analyze microtubule organization in situ in the body wall when investigating mechanisms that control tissue and cell shape.

Immunohistological Labeling of Microtubules in Sensory Neuron Dendrites, Tracheae, and Muscles in the Drosophila Larva Body Wall

To understand how complex cell shapes, such as neuronal dendrites, are achieved during development, it is important to be able to accurately assay microtubule organization. Here we describe a robust immunohistological labeling method to examine microtubule organization of dendritic arborization neuron sensory dendrites, trachea, muscle, and other Drosophila larva body wall tissues.

免疫組織学的な感覚ニューロンの樹状突起における微小管の標識、気管、との筋肉ショウジョウバエ幼虫の体壁

To understand how differences in complex cell shapes are achieved, it is important to accurately follow microtubule organization. The Drosophila larval ...

神経科学

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

郭清ときのこ体の免疫蛍光染色と大人のショウジョウバエ脳光受容器ニューロン

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Live Imaging of Drosophila Larval Neuroblasts

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given their relevance to development and disease, understanding the mechanisms that govern asymmetric stem cell division has been a robust area of study. Because they are genetically tractable and undergo successive rounds of cell division about once every hour, the stem cells of the Drosophila central nervous system, or neuroblasts, are indispensable models for the study of stem cell division. About 100 neural stem cells are located near the surface of each of the two larval brain lobes, making this model system particularly useful for live imaging microscopy studies. In this work, we review several approaches widely used to visualize stem cell divisions, and we address the relative advantages and disadvantages of those techniques that employ dissociated versus intact brain tissues. We also detail our simplified protocol used to explant whole brains from third instar larvae for live cell imaging and fixed analysis applications.

Live Imaging of Drosophila Larval Neuroblasts

This protocol details a streamlined method used to conduct live cell imaging in the context of an intact larval brain. Live cell imaging approaches are invaluable for the study of asymmetric neural stem cell divisions as well as other neurogenic and developmental processes, consistently uncovering mechanisms that were previously overlooked.

のライブイメージング<em&gt;ショウジョウバエ</em&gt;幼虫の神経芽細胞

Stem cells divide asymmetrically to generate two progeny cells with unequal fate potential: a self-renewing stem cell and a differentiating cell. Given ...

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.

Electrophysiological Recording of The Central Nervous System Activity of Third-Instar  Drosophila Melanogaster

This protocol describes a method to record the descending electrical activity of the Drosophila melanogaster central nervous system to enable the cost-efficient and convenient testing of pharmacological agents, genetic mutations of neural proteins, and/or the role of unexplored physiological pathways.

第三令キイロショウジョウバエの中枢神経活動の電気生理学的記録

The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield ...

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these mechanisms is crucial for developing neural regenerative therapies. Drosophila melanogaster is a leading model for studies of neural development but historically has not been exploited to investigate adult brain regeneration. This is primarily because the adult brain exhibits very low mitotic activity. Nonetheless, penetrating traumatic brain injury (PTBI) to the adult Drosophila central brain triggers the generation of new neurons and new glia. The powerful genetic tools available in Drosophila combined with the simple but rigorous injury protocol described here now make adult Drosophila brain a robust model for neural regeneration research. Provided here are detailed instructions for (1) penetrating injuries to the adult central brain and (2) dissection, immunohistochemistry, and imaging post-injury. These protocols yield highly reproducible results and will facilitate additional studies to dissect mechanisms underlying neural regeneration.

Stimulating and Analyzing Adult Neurogenesis in the Drosophila Central Brain

This article provides detailed protocols for inflicting Penetrating Traumatic Brain Injury (PTBI) to adult Drosophila and examining the resulting neurogenesis.

ショウジョウバエ中枢脳における成人神経新生の刺激と分析

The molecular and cellular mechanisms underlying neurogenesis in response to disease or injury are not well understood. However, understanding these ...

Retrograde Labeling of Drosophila Motor Neurons Using Fluorescent Lipophilic Dyes

<a href='https://appjove.unicartagenaproxy.elogim.com/v/60716/retrograde-tracing-drosophila-embryonic-motor-neurons-using'>Source: Inal, M. A. et. al., Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes. J. Vis. Exp. (2020)</a>This video demonstrates the method of retrograde labeling of Drosophila motor neurons using lipophilic dyes. The dye enters the axon, travels retrogradely, and labels the neurons green, allowing detailed visualization of their morphology and projections under a confocal microscope.

Source: Inal, M. A. et. al., Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes. J. Vis. Exp. (2020)This video ...

の中枢神経系における単一細胞のラベリング

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