Name: a method for characterizing embryogenesis in arabidopsis
Uploaded: 2017-08-04T10:00:00.000+00:00
Duration: 10 min 24 s
Description: Watch this Scientific Journal Video about A Method for Characterizing Embryogenesis in Arabidopsis at JoVE.com

博士ジェシカ Habashi は、批判的に読む原稿を感謝いたします。私たちはDR5 GFPの試金のローカリゼーションを実行するイメージング センター、ライフ サイエンス、首都師範大学 (中国北京) の大学の博士 Xianyong 盛を感謝します。この作品は北京市政府科学財団からの助成金によって支えられた (CIT &#38; TCD20150102)、中国の国家自然科学基金 (31600248) から。

注: このプロトコルは 3 つの部分: 1) DIC 顕微鏡を用いた野生型シロイヌナズナ胚のパターン形成の観察2)、胚パターン形成を介して共焦点レーザー走査顕微鏡を用いたマーカー蛋白質発現の観察評価・ 3) (例としてnaa10変異体を使用して) 胚発生における特定遺伝子の役割を決定します。
1. 胚珠クリア
<ol>
	<li>植物材料の準備 注: ここで説明されているプロトコルは健康な植物を育てる方法の例健康な植物を育てるための他の方法はまた働きます。<ol>
		<li>1.5 mL チューブに 100 種を追加し、消毒 5 分反転種子チューブ数回種子とソリューションが十分に混合された表面の種子を完全に殺菌することを確保するために 1.5% ナトリウム次亜塩素酸 (ミックス 85 mL の水で 10% 塩素 15 mL) の 1 つの mL を追加します。</li>
		<li>チューブの下に 30 の遠心分離によって種子を強制的に 1,200 x g で s が、上澄みを除去し、残留ナトリウム次亜塩素酸を削除するベンチで 4 回滅菌水で種を洗います。</li>
		<li>4.4 g 培地、培 (MS) 基底塩の混合物とショ糖の 10 g を純水 1 L に追加します。塩とスクロースを分解する混合物をかき混ぜなさい。MS のショ糖にゲル化剤、Phytagel などの 3 g を追加 ph 5.8 1 m コ、ソリューションを調整し、121 ° C、15 分 MS 培地で溶液を滅菌します。</li>
		<li>MS 培地 30 mL を MS 培地プレートを生成する 12 cm 培養皿に注ぐ。</li>
		<li>MS 板に種子を滅菌を置きます。プレートを生育室に転送して、シード成層 (暗闇の中の 4 ° C で 3 日) 後長日条件 (16 h、光と暗闇の中で 8 h の 18 ° C の 22 ° C) の下でプレート。</li>
		<li>成長の 8 日後のトレイに土 (ミックス栄養豊富な土とバーミキュ ライト 1:1 の比率で) に植物を転送、水損失を防ぐために 5 日間の白、透明な蓋付きトレイを蓋をして植物のガラス化を避けるためにギャップを残します。</li>
		<li>トレイのふたを取るし、長日条件 (1.1.5 参照) の下でウォークイン チャンバー内で植物を栽培します。6:00 に光をオンし、オフで 22:00。ウォークインの商工会議所での成長の約 20 日の間、植物が開花して siliques を生産します。</li>
		<li>花蕾での種の位置にスレッドは 20:00 花房内の茎マーク (最初の 3 番目の花のつぼみには使用しないでください; 通常これらのつぼみから生成された siliques は少数の中止された種子を開発し、異常胚珠に通常の割合を変更この可能性があります)。8:00 次の朝に花を開くとマークの芽として胚珠の受粉の先頭を定義します。</li>
	</ol>
	</li>
	<li>ホイヤーの溶液の調製
	<ol>
		<li>50 mL チューブに抱水クロラールの 24 g を追加し、(抱水クロラールのソリューションは、光の下で安定しておりません) アルミ箔で完全にチューブをラップします。</li>
		<li>次に、上記のソリューションに 9 mL の純水とグリセリンの 3 mL を追加し、抱水クロラールを溶解する管を振る。ホイヤーのソリューションをするためには、任意の気泡を除去するために室温で一晩放置チューブをしましょう。</li>
	</ol>
	</li>
	<li>胚珠の分離とのクリアランス
	<ol>
		<li>顕微鏡のスライド ガラスに両面粘着テープの部分を接続します。表面に垂直な pseudoseptum とスライドの受粉 (DAP) 日後の所望の数の成長 silique を場所します。心皮 (図 1 aと1 b) を分割し、やすくなります。</li>
		<li>注射針、pseudoseptum の両側の心皮を分割し、silique の胚珠が (図 1と1 D) 万国実体写真の下に表示されるようにスライドに 2 つの解剖の心皮を添付します。</li>
		<li>スライド ガラスにホイヤーの液の 70 μ L を分注、浸すことによってホイヤーのソリューションを先の細いピンセットの先端を湿らせてください。使用して、万国実体写真、移動胚珠 Hoyer のソリューションにスライドに胚をきれいにピンセットで。任意の気泡を生成を避けるためにゆっくりスライドする観察を適用します。</li>
		<li>室温で (より成熟した胚珠より多くの時間が必要) 2-12 h の暗い部屋のボックスに完成したスライドを置きます。(1-2 DAP) 2/4 細胞期胚は、4 h 後八分と球状初期 (3 DAP) 間の胚を観察できる中 2 h 後顕微鏡で観察できます。12 時間後より成熟胚 (4-8 DAP) が観察できます。</li>
		<li>顕微鏡の DIC 関数を使用してクリアされた胚珠を調べます。低消費電力フィールド (10 X) の下で胚を見つけ、開発の胚の細胞のパターンを観察する高出力フィールド (100 倍油浸漬レンズ) に変更します。</li>
		<li>発達段階別に胚を確認します。</li>
	</ol>
	</li>
</ol>
2. 胚パターン形成と細胞マーカー発現の観測によるアイデンティティのキャラクタリゼーション
<ol>
	<li>植物材料の準備
	<ol>
		<li>プロモーターとマーカー遺伝子やホルモンの応答の要素のシーケンスを符号化を複製し、( GFP) などの蛍光タンパク質を融合次に、バイナリのベクター (pCambia1300) などに構成要素を挿入します。ここでは、オーキシン応答要素DR5は、例10として表示されます。</li>
		<li>アグロバクテリウムによる野生型植物に結果のDR5 GFPプラスミドを導入-変換11を介する。MS 培地プレート hygromycin T1 植物を選ぶ (50 mg/mL 母液の希釈 1:1, 000)。遺伝子組換え植物は、長い根を展示、1.1.5 に記載されている条件の下で成長の 8 日後の 2 つのロゼット葉を生成します。</li>
		<li>遺伝子組換え植物を土に転送、1.1.6 1.1.7 で示されているように、それらを養います。</li>
		<li>独立した t1 から T2 の種子を収集します。MS hygromycin 板に種をまく、 DR5 GFPの挿入 (敏感な植物に抵抗力がある植物の比になる約 3:1) の 1 つのコピーを運ぶ植物を選択し、土壌にそれらを転送します。</li>
		<li>1.1.6 1.1.7 で示されているように、T2 植物を栽培します。</li>
		<li>T2 植物から T3 種子を収集します。MS hygromycin 板に種をまく、ホモ接合体の遺伝子組換え植物 (hygromycin 抵抗を展示、すべての単一の行からの植物) を選択し、土壌にそれらを転送します。</li>
		<li>1.1.8 で示されているように、ホモの T3 植物の花蕾をマークします。</li>
	</ol>
	</li>
	<li>GFP 信号の観察
	<ol>
		<li>47 ml の 6% グリセリン溶液を生成する超純水のグリセロールの 3 mL を混ぜます。</li>
		<li>両面粘着テープの一部をガラス顕微鏡スライドに添付し、1.3.1 に示すように、silique を修正します。</li>
		<li>注射針で、pseudoseptum の両側の心皮を分割、1.3.2 に示すよう堅実下スライドに 2 つの解剖の心皮を取り付けます。</li>
		<li>スライド ガラスの上に 6% のグリセロールの 30 μ L を分注、先の細いピンセットを使用してソリューションに切り裂かれた心皮から胚珠のすべてを配置します。スライドにそっと観察を配置します。</li>
		<li>種皮と胚乳それぞれの胚珠には、胚の GFP の信号の観測を防ぐことができます、胚胚珠から抽出します。迅速かつ慎重にスライドをタップ (より成熟した胚珠は、スライドに適用する以下の力) 胚珠から胎児を押し出す。</li>
		<li>GFP 蛍光共焦点レーザー顕微鏡のスライドを確認します。低消費電力フィールド (10 X) の下で分離胚を見つけるとその場所を記録し、488 に励起光源を設定することによって胚におけるDR5 GFP発現を観察する高出力フィールド (100 倍油浸漬レンズ) に変更 GFP 信号の検出のための nm。</li>
		<li>さらに分析に適した T3 ラインを選択する胚の GFP 発現を検出します。</li>
	</ol>
	</li>
</ol>
3 胚珠クリアランスとマーカー蛋白質観察で胚致死突然変異体の胚パターン形成の物理特性: 例としてNaa10
<ol>
	<li>Naa10突然変異体植物の胚パターン形成
	<ol>
		<li>1.1.1-1.1.7 に示されているように、植物材料を準備します。</li>
		<li>Naa10を識別する+/−植物遺伝子特定のプライマーの5としてマークを用いた PCR によるなぁ10 の花蕾+/− 1.1.8 に記載されている植物します。</li>
		<li>胚珠のクリアランスとなぁ10 の顕微鏡検査を実行+/−胚 1.2 と 1.3 で示される。</li>
	</ol>
	</li>
	<li>Naa10胚を使用してマーカー蛋白質解析
	<ol>
		<li>2.1 2.2 で説明されている手順を使用して適切なDR5 GFPトランスジェニックは行を取得します。</li>
		<li>ホモなのなぁ10 DR5 GFPトランスジェニック一線を越え+/− なぁ10 を取得する工場+/− naa10を特徴付ける (遺伝子特定の PCR で識別) F2 世代で GFP 蛍光マーカーのホモ接合体である植物+/−5ホモDR5 GFPラインを使用して顕微鏡下で GFP シグナルを観察し、。</li>
		<li>なぁ10 の花芽をマーク+/− DR5 GFP 2.1.7 に示すようのホモ接合体である植物。</li>
		<li>DR5 GFP 2.2 に示すようにホモがnaa10胚の GFP の信号を検索します。</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Jenik, P. D., Gillmor, C. S., Lukowitz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+patterning+in+Arabidopsis+thaliana.">Embryonic patterning in Arabidopsis thaliana.</a> Annu Rev Cell Dev Biol. 23, 207-236 (2007).</li><li>Lau, S., Slane, D., Herud, O., Kong, J., Jurgens, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+embryogenesis+in+flowering+plants:+setting+up+the+basic+body+pattern.">Early embryogenesis in flowering plants: setting up the basic body pattern.</a> Annu Rev Plant Biol. 63, 483-506 (2012).</li><li>Wendrich, J. 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胚珠のクリアランスは、細胞分裂を検出およびシロイヌナズナの胚の形態を評価するための便利なメソッドこの手法を使用して、胚が DIC 顕微鏡5,12の下で直接観察できます。胚珠のクリアランスのための重要なステップは、1.3.4 のステップです。1.3.4 の手順で胚珠クリアランスに必要な時間変数です。2/4 細胞期胚はホイヤーのソリューショ?...

胚より高い植物の開発の最も早いイベントです。細胞分裂と分化厳格な遺伝子管理1,2の下で受精卵から胚形態。シロイヌナズナの胚は、胚発生の間に細胞分裂の順序が予想されるパターンは3,4あるため形態形成の制御の研究に有用なモデルです。ただし、複数のセルを 1 つを含むシロイヌナズナ胚は観察して分析するのには小さすぎます。さらに、ある特定の遺伝子の突然変異は胚致死5胚のそれらの遺伝子の重要な役割を示す可能性があります。胚致死突然変異体におけるパターン形成の解析という必須遺伝子は胚発生を調節する分子機構を理解するための基礎を提供します。
詳細な方法は、直接顕微鏡観察によってモデル植物シロイヌナズナの胚パターン形成の特性評価のためここで記述されます。メソッドには、胎児の観察に続いて胚珠クリアランスが含まれます。胚致死突然変異体の特性評価についても述べる.
さまざまな発達段階で胚の形態は、ホイヤーのソリューション6,7でクリアされている胚珠を使用して顕微鏡による直接決定できます。このような胚珠展示高屈折;したがって、メソッドをクリアこの胚珠は差動干渉の対照 (DIC) 顕微鏡で観察する必要があります標本で特に有利です。
さらに、胚発生時に特定のセルまたはセルの限られた数で表現される遺伝子は、胚の細胞を識別するマーカーとして使用できます。したがって、共焦点レーザ走査型顕微鏡を使用してマーカーの GFP 付けられた蛋白質の発現を監視することによって胚発生の間に細胞の identity の指定を付けることができます。逆に、胚発生中に不明な機能遺伝子 -GFP融合の発現パターンを観察し、胚のパターン形成とその遺伝子の発現の間のリンクを提供できますシロイヌナズナの胚発生に必要な新しい遺伝子の同定を容易にします。
ここで説明する方法は、胚致死性の変異体の表現型を特徴付けると胚細胞レベルでの影響を受けた遺伝子の影響を判断するにも使えます。さらに、野生型と変異型の植物間の胚発生中にマーカー遺伝子の発現パターンの比較と組み合わせて、メソッドは胚8,9に関与するシグナル伝達経路の分子機構についての手がかりを得ることができます。確かに、 naa10の植物に適用した、変異における脳下垂体の異常分裂が胚5中のオーキシンの分布の変化によって引き起こされることが分かった。

<table><tbody><tr><td>Chloral Hydrate</td><td>Sigma</td><td>15307</td><td></td></tr><tr><td>Murashige and Skoog Basal Medium</td><td>Sigma</td><td>M5519</td><td></td></tr><tr><td>Phytagel</td><td>Sigma</td><td>P8169</td><td></td></tr><tr><td>Hygromycin</td><td>Roche</td><td>10843555001</td><td></td></tr><tr><td>Growth chamber</td><td>Percival</td><td>CU36L5</td><td></td></tr><tr><td>Fluorescence microscope</td><td>Zeiss Oberkochen</td><td>Zeiss Image M2</td><td>camera: AxioCam 506 color</td></tr><tr><td>Confocal Laser Scanning Microscopy</td><td>Zeiss Oberkochen</td><td>Zeiss LSM 5</td><td>filters: BP 495 - 555 nm</td></tr><tr><td>Stereoscope</td><td>Zeiss Oberkochen</td><td>Axio Zoom V16</td><td>camera: AxioCam MRc5</td></tr><tr><td>nutrient-rich soil</td><td>KLASMANN, Germany</td><td></td><td></td></tr><tr><td>50-mL tube</td><td>Corning Inc.</td><td></td><td></td></tr><tr><td>1.5-mL tube</td><td>Corning Inc.</td><td></td><td></td></tr><tr><td>10% sodium hypochlorite solution</td><td></td><td></td><td>Domestic analytical reagent</td></tr><tr><td>sucrose</td><td></td><td></td><td>Domestic analytical reagent</td></tr><tr><td>KOH</td><td></td><td></td><td>Domestic analytical reagent</td></tr><tr><td>glycerol</td><td></td><td></td><td>Domestic analytical reagent</td></tr><tr><td>culture dish</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>vermiculite</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>glass slide</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>syringe needle</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>fine-tipped tweezers</td><td></td><td></td><td>Domestic supplies</td></tr><tr><td>super clean bench</td><td></td><td></td><td>Domestic equipment</td></tr></tbody></table>

a method for characterizing embryogenesis in arabidopsis

彼らの開発の高い予測可能な性質を与えられて、シロイヌナズナ胚として使用されている、モデル植物の形態形成の研究。ただし、早い段階の植物の胚は小さく、観察・分析が困難、ほとんどの細胞を含みません。モデル生物のシロイヌナズナを用いた顕微鏡下で植物胚におけるパターン形成の手法が記載されています。ホイヤーのソリューションを使用して新鮮な胚珠のクリアランス、胚の形態と細胞数の観察、発達段階には、微分干渉顕微鏡倍油浸漬レンズ 100 を使用してによって決められるかもしれません。また、タグ付きの緑色蛍光タンパク質 (GFP) と特定のマーカー蛋白質の表現は、共焦点レーザ走査型顕微鏡により胚形成の間にセル identity の指定に注釈を付ける監視だった。したがって、細胞・分子レベルでは、野生型植物胚におけるパターン形成を観察して野生型植物胚致死突然変異胚におけるパターン形成を比較することによって胚パターン形成における特定遺伝子の役割を特徴付けるには、このメソッドを使用できます。したがって、メソッドは、シロイヌナズナの胚発生の特性評価に使用できます。

直接顕微鏡下で胚を観察し胚発生の間に細胞の identity の指定特定のマーカーにコメントを付ける、胚形成の間に特定の遺伝子の役割を特徴付けるこのペーパーで説明する方法を使用できます。
胚 (成熟した棒を歩いてステージに細長い受精段階) から野生型シロイヌナズナにおけるパターン形成の解析から代表の結...

Watch this Scientific Journal Video about A Method for Characterizing Embryogenesis in Arabidopsis at JoVE.com

A Method for Characterizing Embryogenesis in Arabidopsis

このプロトコルでは、顕微鏡下で胚パターン形成の検査続く胚珠クリアランスを介してシロイヌナズナの胚発生を観察するためのメソッドについて説明します。

シロイヌナズナの胚発生の特性評価法

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

a-method-for-characterizing-embryogenesis-in-arabidopsis

Universidad San Sebastian

Research

JoVE Journal

Developmental Biology

A Method for Characterizing Embryogenesis in Arabidopsis

11.1K ビュー.  Capital Normal University. このプロトコルでは、顕微鏡下で胚パターン形成の検査続く胚珠クリアランスを介してシロイヌナズナの胚発生を観察するためのメソッドについて説明します。

ビデオ: A Method for Characterizing Embryogenesis in Arabidopsis

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

モデルマメに初期胚発生の調節を研究するための接合体と体細胞胚を得るためのプロトコル<em&gt;のMedicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

発生生物学

Live Confocal Imaging of Developing Arabidopsis Flowers

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. Recent advances in confocal microscopy, combined with the development of numerous fluorophores, have overcome these issues and opened the possibility to study the expression of several genes simultaneously, with a good cellular resolution, in live samples. Live confocal imaging provides plant biologists with a powerful tool to study development, and has been extensively used to study root growth and the formation of lateral organs on the flanks of the shoot apical meristem. However, it has not been widely applied to the study of flower development, in part due to challenges that are specific to imaging flowers, such as the sepals that grow over the flower meristem, and filter out the fluorescence from underlying tissues. Here, we present a detailed protocol to perform live confocal imaging on live, developing Arabidopsis flower buds, using either an upright or an inverted microscope.

Live confocal imaging provides biologists with a powerful tool to study development. Here, we present a detailed protocol for the live confocal imaging of developing Arabidopsis flowers.

共焦点イメージングは​​、シロイヌナズナの花の開発のライブ

The study of plant growth and development has long relied on experimental techniques using dead, fixed tissues and lacking proper cellular resolution. ...

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; for example, how the zygote polarizes to establish the body axis and how the embryo specifies various cell fates during organ formation. This manuscript describes an in vitro ovule culture method to perform live-cell imaging of developing zygotes and embryos of Arabidopsis thaliana. The optimized cultivation medium allows zygotes or early embryos to grow into fertile plants. By combining it with a poly(dimethylsiloxane) (PDMS) micropillar array device, the ovule is held in the liquid medium in the same position. This fixation is crucial to observe the same ovule under a microscope for several days from the zygotic division to the late embryo stage. The resulting live-cell imaging can be used to monitor the real-time dynamics of zygote polarization, such as nuclear migration and cytoskeleton rearrangement, and also the cell division timing and cell fate specification during embryo patterning. Furthermore, this ovule cultivation system can be combined with inhibitor treatments to analyze the effects of various factors on embryo development, and with optical manipulations such as laser disruption to examine the role of cell-cell communication.

In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana

This manuscript describes an in vitro ovule cultivation method that enables live-cell imaging of Arabidopsis zygotes and embryos. This method is utilized to visualize the intracellular dynamics during zygote polarization and the cell fate specification in developing embryos.

体外受精卵の Live 細胞イメージング偏光とシロイヌナズナの胚パターン形成の胚珠培養

In most flowering plants, the zygote and embryo are hidden deep in the mother tissue, and thus it has long been a mystery of how they develop dynamically; ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Translating Ribosome Affinity Purification (TRAP) to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome affinity purification (TRAP) method and consecutive optimized low-input library preparation.
As starting material, we employ plant lines that express GFP-tagged ribosomal protein RPL18 in a cell type-specific manner by use of adequate promoters. Prior to immunopurification and RNA extraction, the tissue is snap frozen, which preserves tissue integrity and simultaneously allows execution of time series studies with high temporal resolution. Notably, cell wall structures remain intact, which is a major drawback in alternative procedures such as fluorescence-activated cell sorting-based approaches that rely on tissue protoplasting to isolate distinct cell populations. Additionally, no tissue fixation is necessary as in laser capture microdissection-based techniques, which allows high-quality RNA to be obtained.
However, sampling from subpopulations of cells and only isolating polysome-associated RNA severely limits RNA yields. It is, therefore, necessary to apply sufficiently sensitive library preparation methods for successful data acquisition by RNA-seq.
TRAP offers an ideal tool for plant research as many developmental processes involve cell wall-related and mechanical signaling pathways. The use of promoters to target specific cell populations is bridging the gap between organ and single-cell level that in turn suffer from little resolution or very high costs. Here, we apply TRAP to study cell-cell communication in lateral root formation.

Translating Ribosome Affinity Purification TRAP to Investigate Arabidopsis thaliana Root Development at a Cell Type-Specific Scale

Translating ribosome affinity purification (TRAP) offers the possibility to dissect developmental programs with minimal processing of organs and tissues. The protocol yields high-quality RNA from cells targeted with a green fluorescent protein (GFP)-labeled ribosomal subunit. Downstream analysis tools, such as qRT-PCR or RNA-seq, reveal tissue and cell type-specific expression profiles.

細胞型特異的スケールでのシロイヌナズナ・タリアナの開発を調査するためのリボソーム親和性精製(TRAP)の翻訳

In this article, we give hands-on instructions to obtain translatome data from different Arabidopsis thaliana root cell types via the translating ribosome ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

生物学

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

の長期、高分解能共焦点タイムラプスイメージング<em&gt;シロイヌナズナ子葉</em発芽過程&gt;表皮

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

から初期段階の胚の効率的かつ迅速な単離<em&gt;シロイヌナズナ</em&gt;種子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

ホールマウント胚珠内でのクロマチン修飾や核構造の定量的、単一細胞分析のための効率的な方法<em&gt;シロイヌナズナ</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant productivity and survival. As such, understanding the mechanisms by which stomata develop and pattern has tremendous agronomic value. This paper describes two phenotypic methods using Arabidopsis cotyledons that can be used to characterize the genes controlling stomatal development and patterning. Presented first are procedures for analyzing the stomatal phenotypes using toluidine blue O-stained cotyledons. This method is fast and reliable and does not require the use of epidermal peels, which are widely used for phenotypic analyses but require specialized training. Due to the presence of multiple cysteine residues, the identification and generation of bioactive EPF peptides that have a role in stomatal development have been challenging. Thus, presented second is a procedure used to identify stomatal ligands and monitor their biological activity by bioassays. The main advantage of this method is that it produces reproducible data relatively easily while reducing the amount of peptide solution and the time required to characterize the role of the peptides in controlling stomatal patterning and development. Overall, these well-designed protocols enhance the efficiency of studying the potential stomatal regulators, including cysteine-rich secretory peptides, which require highly complex structures for their activity.

Identification of the Genes Involved in Stomatal Development via Epidermal Phenotype Scoring

This paper describes two phenotyping methods without the use of epidermal peels to characterize the genes controlling stomatal development. The first method demonstrates how to analyze a stomatal phenotype using a toluidine blue O-stained plant epidermis. The second method describes how to identify stomatal ligands and monitor their biological activities.

表皮表現型スコアリング による 気孔発生に関与する遺伝子の同定

Stomata are small pores on the surface of land plants that are involved in gas exchange and water vapor release, and their function is critical for plant ...

シロイヌナズナの胚発生の特性評価法

要約

さらに動画を探す

この動画の章

モデルマメに初期胚発生の調節を研究するための接合体と体細胞胚を得るためのプロトコル

共焦点イメージングは、シロイヌナズナの花の開発のライブ

体外受精卵の Live 細胞イメージング偏光とシロイヌナズナの胚パターン形成の胚珠培養

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

細胞型特異的スケールでのシロイヌナズナ・タリアナの開発を調査するためのリボソーム親和性精製(TRAP)の翻訳

でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ

の長期、高分解能共焦点タイムラプスイメージング

から初期段階の胚の効率的かつ迅速な単離

ホールマウント胚珠内でのクロマチン修飾や核構造の定量的、単一細胞分析のための効率的な方法

表皮表現型スコアリングによる気孔発生に関与する遺伝子の同定

シロイヌナズナの胚発生の特性評価法

要約

さらに動画を探す

この動画の章

モデルマメに初期胚発生の調節を研究するための接合体と体細胞胚を得るためのプロトコル

共焦点イメージングは​​、シロイヌナズナの花の開発のライブ

体外受精卵の Live 細胞イメージング偏光とシロイヌナズナの胚パターン形成の胚珠培養

C.エレガンス胚発生を解析するセミハイスループットイメージング法とデータ可視化ツールキット

細胞型特異的スケールでのシロイヌナズナ・タリアナの開発を調査するためのリボソーム親和性精製(TRAP)の翻訳

でFACSアシスト細胞型特異的発現プロファイリングを使用して組織と器官特異フィトクロム応答を調査シロイヌナズナ

の長期、高分解能共焦点タイムラプスイメージング

から初期段階の胚の効率的かつ迅速な単離

ホールマウント胚珠内でのクロマチン修飾や核構造の定量的、単一細胞分析のための効率的な方法

表皮表現型スコアリング による 気孔発生に関与する遺伝子の同定

共焦点イメージングは、シロイヌナズナの花の開発のライブ

表皮表現型スコアリングによる気孔発生に関与する遺伝子の同定