Name: single-cell photoconversion in living intact zebrafish
Uploaded: 2018-03-19T14:00:00.000+00:00
Duration: 9 min 49 s
Description: Watch this Scientific Journal Video about Single-cell Photoconversion in Living Intact Zebrafish at JoVE.com

ベルナール ・ Kulemaka とサム ・ コネルとブレント レッドフォード 3i ゼブラフィッシュ ケアのため画像の質問とデボラ ビッグバン カレン耳を傾ける、ケイ ・ スチュワートの守備のための有用なコメントと試薬指導、スミス研究室のメンバーに感謝しますこの作品は、ゼブラフィッシュ研究幹細胞のセンターとノートルダム大学とノートルダム大学で再生医療のノートルダム大学、エリザベスとマイケル ・ ギャラガー家族、アルフレッド ・ p. スローン財団、センターによって支えられました。大聖堂。すべての動物の研究は、博士コーディ スミスに IACUC ノートルダム大学に準拠して行われました。

すべての動物の研究は、ノートルダム大聖堂機関動物ケアおよび使用委員会の大学によって承認されました。
1. ゼブラフィッシュ標本の作製
<ol><li>標準的な手順13あたり交尾商工会議所にオス一匹と 1 つアダルト女性 Tg (転換タンパク質) を配置します。本稿では、Tg(sox10:eos) 魚9アクセスが蛍光蛋白質と他の形質転換線を同様に使用することができますを使用します。魚では、配置を行う場合に 1 つ以上の区域を設定します。一晩部屋のままに魚を許可します。</li>
	<li>次の朝は、100 mm のペトリ皿に卵を収集します。上映前に卵を成熟させ 24 時間後受精 (hpf) を許可します。</li>
	<li>上記の動物の遺伝子のヘテロ接合体なら、Tg(sox10:eos) + 488 nm 光源と GFP と胚の画面 24 hpf 料理は解剖顕微鏡のセットをフィルターします。Tg(sox10:eos) + 胚を分離でき、48 に成熟する hpf。</li>
	<li>針やピンセットを使って手動で Dechorionate 胚。</li>
	<li>準備、0.8% 低溶解点アガロース溶液の 5 mL を電子レンジします。<ol>
<li>Agarose が touch にクール、場所 3-4 麻酔 Tg (sox10:eos) +48 10 mm のガラス基板の中央に hpf 魚底シャーレ。</li>
		<li>カバーガラス、約 1 mL の表面をカバーする十分な agarose を追加します。探針を使用すると、その両側に魚を配置できます。動物の取付を確実に固めるための agarose を許可します。寒天の凝固14まで、ゼブラフィッシュを継続的に再配置する必要があります。凝固は約 2 分です。</li>
	</ol>
</li>
	<li>2 分の agarose が凝固した後、ゆっくりと胚培 0.02% アミノ安息香酸エステル (Tricaine) 寒天や皿の底面を水中まで皿を追加します。これは、約 3 mL をする必要があります。</li>
</ol>2. 顕微鏡取り付けと事前変換イメージング
<ol><li>共焦点ソフトウェアを開き、キャプチャ、フォーカス ウィンドウ [図 1] を選択します。キャプチャ ウィンドウの下には、[キャプチャ設定] タブ [図 1] ドロップダウン ラボ固有の変換イメージング設定を選択します。ここでは、ラボ固有の設定「魚イメージングします」と呼びます</li>
	<li>共焦点範囲に試験片を置き、コースと微調整ノブを使用してフォーカスに。</li>
	<li>フォーカス ウィンドウを開き、興味 (すなわち後根神経節) の目的の地域を探します。</li>
	<li>C488 レーザー フィルターを設定] メニューの [を選択します。300 ms に露出を設定、5、パワー、75 [図 2] に強化します。</li>
	<li>キャプチャの種類] セクションの [3 D] ボックスを確認します。[ 3 D キャプチャ] セクションで、現在の位置を使用してを選択し、電流のまわりの範囲をチェックします。同じセクションの範囲を 35、36、飛行機と 1 ステップ サイズの数に設定します。範囲を増加またはイメージング領域の深さに対応して縮小することができます。脊髄の間 35-40 スタック範囲の値は通常、[図 5] は。</li>
	<li>[図 5] の現在の場所を選択します。</li>
	<li>画像を取得するには、[キャプチャ] ウィンドウの下部に [開始。</li>
</ol>3. 単一セルの光電
<ol><li>共焦点ソフトウェアを開き、キャプチャ、フォーカス ウィンドウ [図 1] を選択します。キャプチャ ウィンドウの下には、[キャプチャ設定] タブ [図 1] ドロップダウン ラボ固有の変換イメージング設定を選択します。ここでは、ラボ固有の設定は「魚は全チップを切除」と呼ばれる</li>
	<li>C488 c541 レーザー フィルターを設定] メニューの [を選択します。同じ顕微鏡のソフトウェアを使用していない場合、は、別のレーザーと選択、488 nm と 541 nm のレーザーを選択するメニューを見つけます。300 ms への露出を設定、5、パワー、75 [図 2] に強化します。これらのレーザーの設定は、退色または毒性を引き起こすことがなく十分な蛍光シグナルを生成に基づいて選択されます。毒性や退色が視覚化されるレーザー パワーや露出を減らします。</li>
	<li>フォーカス ウィンドウを開き、フォーカス ウィンドウの [photomanipulation] タブをクリックします。レーザーのパラメーターを調整します。2、スタックのレーザー出力を変更しをクリックしてGo。ラスター ブロック サイズを 1 に変更し、設定をクリックします。ダブルクリックしてサイズを 4 に変更します。レーザー ラインを v405 に変更 [図 3]。</li>
	<li>キャプチャ ウィンドウの高度なキャプチャ設定を開きます。[Photomanipulation] タブを選択し、ダブルクリックして繰り返しを 2 に変更します。[図 4] [ok] をクリックします。</li>
	<li>フォーカス ウィンドウで、[XY] タブを選択します。[図 3,図 4] の [photomanipulation] タブで手順 2 からレーザーのパラメーターを再確認してください。Photoconvert なく周囲のセルの光電細胞にレーザーの設定を設定します。<ol>
<li>隣接するセルの光電が存在する場合は、レーザー出力を下げます。セルの光電が発生しない場合は、レーザーを増やすことができます。最適な photoconvert 興味のない周辺地域のみにレーザーの出力を設定します。</li>
	</ol>
</li>
	<li>タイムラプスをオンにすると、下の種類をキャプチャします。[図 6A] を開始をクリックします。</li>
	<li>ライブ タイムラプス ウィンドウが開いたら、[図 7] の上部のツールバーで、[円] ツールを選択します。</li>
	<li>セルの中央地域で円を描きます。引かれた円を右クリックして、 FRAP 地域を選択し、3 秒間待ちます。選択した領域は暗くなるべき [図 8]。キャプチャを停止をクリックします。</li>
	<li>1 つ以上の動物をイメージングする場合フォーカス メニューと選択位置 2 [XY] タブに戻ります。手順 4.1 4.6 を繰り返します。</li>
	<li>セルの人口を変換するには、手順 4.1 4.4 FRAP 関心領域は、線ツールを使用して円を描くのではなくのプロトコルおよびレーザーのパラメーターします。関心領域に直線を描くし、上記の同じパラメーターを使用して、領域を FRAP します。</li>
</ol>4. ポスト光電イメージング
<ol><li>すべてのポイントが photoconverted です。Precoversion イメージング手順 3 に記載されている設定ラボ固有の標準画像のスタックを選択します。これは、キャプチャ キャプチャ ウィンドウ [図 5] タブのドロップダウンの設定下にあります。</li>
	<li>C488 レーザーを選択し ms に露出を設定、5、パワー、75 [図 2] に強化します。</li>
	<li>C488 レーザーと同じメニューの下にある c541 レーザーを選択します。500 ms に露出を設定、レーザーの電源を 10、および 75 [図 9] を強化します。</li>
	<li>キャプチャの種類] セクションの [3 D] ボックスを確認します。[ 3 D キャプチャ] セクションで、現在の位置を使用してを選択し、電流のまわりの範囲をチェックします。同じセクションの範囲を 35、36、飛行機と 1 ステップ サイズの数に設定します。増加または [図 5] 希望のイメージング深さに合わせて減少する範囲数。</li>
	<li>XY フォーカス] タブで複数のポイントがある場合は、キャプチャ ウィンドウでマルチポイント リスト オプションを選択します。ない場合は、現在の場所を選択します。</li>
	<li>画像を取得するには、[キャプチャ] ウィンドウの下部に [開始。</li>
</ol>

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複雑な組織の種類セルは特定のドメインに整理します。最近の技術はこれらの大規模な組織の構造の1,2,の3の内で個々 のセルをラベルに利用されています。単一細胞の相互作用および複雑な組織内で細胞人口の相互作用を視覚化する同様に利用できる 2 つの手法を示します。光電技術の利点は、科学者はセルをはっきりと...

複数の異なる細胞が複雑な動物や植物の組織の維持・構築に対話します。これらの細胞は、インターカ レートとティッシュの固定を必要とする高分解能顕微鏡なしの単一セルのレベルで近所の人から区別することは困難。しかし、どのようにこれらの組織フォームを理解、維持、病気になるには、組織内の単一のセルを調べることが不可欠がされて時間をかけて相互作用しています。理想的には、これらの実験は、固定する必要がなく非侵襲的な方法で組織内の単一セルのラベルを必要とします。科学者は今この作業1,2,3、4を達成するために多数の技術を開発しました。
発見とクラ ゲの緑色蛍光タンパク質 (GFP) の実装は、組織環境1の異なるセルのラベルの 1 つの刺激的なアプローチだった。細胞特異的発現プロモーターを使用して、それは遺伝子1のラベルが付いているセルのサブセットを選択することが可能です。また、GFP3,4のユーザー選択式の GFP の発現するウイルスを利用できます。GFP 発現の遺伝的媒介が; 組織における細胞のサブセットの内でユーザーが選択した式を許可しませんが、非常に便利でき、GFP のウイルス発現が有利な侵襲的です。単一細胞および複雑な組織2,でそれらの間の相互作用を視覚化することが可能なっている GFP 誘導体および Brainbow 組織内よりまばら異なる蛍光蛋白質を表現するような巧妙な技術の出現で、5します。 ただし、これらのアプローチがランダムな方法で細胞をラベルします。目的の実験には、単一細胞の可視化や実験者によって定義されているセルの人口が必要とする場合そのため制限されます。そのような実験と区別する単一のセル方式で操作できる遺伝子発現蛍光タンパク質を持っているに有利であろう他の蛍光・非蛍光性の細胞から。
この目標を達成するため、複雑な生体組織内の単一セルの細胞生物学を視覚化は、科学的なコミュニティは、異なる蛍光タンパク質6,7,8の単一セルの光電を使用します。緑から赤の蛍光状態紫外線にさらされたときに移行蛍光蛋白質 (すなわち、eos、楓等) の表現を制御する遺伝子を使用して (488 nm) 光、我々 は蛍光に分類されたからの単一のセルを区別できます。隣人6,7,8。このアプローチは、回折限界領域にレーザー スタックからライトを指示することができます私たちの共焦点の顕微鏡に接続されている装置を利用しています。この手法により、我々 はどちらか単一セルまたはユーザー定義の方法9,10,11より大きい人口ラベルできます。手法は、ウイルス ・ GFP の単一細胞注射に比べて低侵襲です。Photoconvert 末梢神経系と脊髄9,10の腹側に位置する細胞のようなより大きい人口を photoconvert 神経節内の単一細胞ことを示して我々 の概念の証拠として 11,12。彼らの動きや開発中に分化に洞察力を得るためにこれら photoconverted のセル人口 24 h 後、視覚化できます。

<table><tbody><tr><td>&lt;font class=&quot;font5&quot;&gt;Tg(&lt;em&gt;sox10:eos&lt;/em&gt;)&lt;/font&gt;&lt;font class=&quot;font0&quot;&gt; zebrafish animals&lt;/font&gt;</td><td></td><td></td><td>Fish were obtained and crossed from the fish facility at the University of Notre Dame</td></tr><tr><td>100 X 15 mm petri dish</td><td>VWR</td><td>25384-302</td><td></td></tr><tr><td>Embryo medium</td><td></td><td></td><td>Embryo medium is made weekly and provided by the fish facility at the University of Notre Dame containing 5L RO water, 30uL methylene blue, and 200mL salt stock.</td></tr><tr><td>0.8% Low Melting Point Agarose</td><td>dot scientific inc</td><td>9012-36-6</td><td></td></tr><tr><td>35 X 10 mm glass-coverslip bottom petri dish</td><td>Ted Pella Inc.</td><td>14021-20</td><td></td></tr><tr><td>Needle dissecting probe</td><td></td><td></td><td></td></tr><tr><td>0.002% 3-aminobenzoic acid ester (Tricaine)</td><td>Fluka analytical</td><td>A5040-250G</td><td></td></tr><tr><td>Fluorescent Dissecting Microscope with GFP filters</td><td>Zeiss Axiozoom</td><td></td><td></td></tr><tr><td>Confocal microscope with lasers to excite GFP and RFP filter sets</td><td>3i spinning disk confocal</td><td></td><td></td></tr><tr><td>UV light source (laser) for photoconversion</td><td>405 nm laser</td><td></td><td></td></tr><tr><td>Slidebook software</td><td>3i</td><td></td><td></td></tr><tr><td>Methylene blue</td><td>Kordon</td><td></td><td></td></tr></tbody></table>

single-cell photoconversion in living intact zebrafish

動物や植物の組織細胞の異なる集団で構成されます。これらの細胞と組織を維持・構築に時間をかけて対話中断されるとき病気を引き起こすことができます。科学者は、細胞のサブセットに蛍光蛋白質を表現することによって特徴とそのまま組織内でこれらの細胞の自然動態を調査するためのテクニックを開発しました。しかし、時は、実験は時単一細胞または細胞の人口のように、組織内の細胞より選択した可視化する必要があります。科学者はこれを実現し、セルの人口内の単一のセルを可視化する蛍光タンパク質の単一セルの光電を活用しています。このテクニックを示すためには、方法を示しますここをそのまま、興味の Eos を表現する細胞に UV ライトを指示するゼブラフィッシュの生活します。私たちは、どのように彼らは組織の変更を確認するそれらの photoconverted Eos+セル 24 時間後をイメージします。二つの手法: 単一セルの光電とセルの人口の photoconversions。これらの技術は、細胞間相互作用、細胞運命と分化、細胞移動、作りそれ多数の生物的質問に適用する技術を視覚化する使用できます。

組織6内の個別のセルにラベルを付けるには、蛍光タンパク質の光変換を使用できます。これを示すには、 Tg(sox10:eos)魚9 sox10の規制のシーケンス下の蛍光蛋白質 Eos を表現する使用されました。48 Tg(sox10:eos)動物 hpf が最初にマウントされていると、可能性があります任意の非特異的光電検出イメージを作成します?...

Watch this Scientific Journal Video about Single-cell Photoconversion in Living Intact Zebrafish at JoVE.com

Single-cell Photoconversion in Living Intact Zebrafish

ここでは、蛍光蛋白質、Eos、生きている動物の表現する特定の領域に紫外線暴露により光電セルを達成する方法を表示するためのプロトコルを提案する.

生活そのままゼブラフィッシュの単一セルの光電

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

single-cell-photoconversion-in-living-intact-zebrafish

Research

JoVE Journal

Developmental Biology

6.0K ビュー.  University of Notre Dame. ここでは、蛍光蛋白質、Eos、生きている動物の表現する特定の領域に紫外線暴露により光電セルを達成する方法を表示するためのプロトコルを提案する.

ビデオ: Single-cell Photoconversion in Living Intact Zebrafish

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

光変換後の蛍光減衰を使用して生活ゼブラフィッシュ胚におけるタンパク質の安定性の測定（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

発生生物学

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

リビングゼブラフィッシュ胚における細胞内構造のイメージング

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Photoconvertible PSmOrangeシステムを用いたGFPトランスジェニックゼブラフィッシュで細胞を追跡

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

多光子顕微鏡によるライブセルイメージングのための大人のトランスジェニックゼブラフィッシュ網膜外植片の文化

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

ゼブラフィッシュ胚における細胞光電を介して次の心内膜組織運動

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

文化、ゼブラフィッシュ初代培養細胞のトランスフェクション

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

ライブゼブラフィッシュ胚における二光子ベースの光活性化

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

生物学

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

トランスジェニックゼブラフィッシュの多色タイムラプスイメージング：標的神経細胞のアブレーションによって活性化を可視化網膜幹細胞

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

ゼブラフィッシュにおける単一の細胞運命のマッピング

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

ゼブラフィッシュの開発時Photoconvertibleタンパク質を用いた細胞トラッキング

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...