この作品での顕微鏡で、研究所のトランスフォーマティブ生命分子 (WPI ITbM) 名古屋大学を行われ、日本高等植物科学ネットワークでサポートされています。この作品は、振興会の日本科学技術振興機構 (ERATO プロジェクト ノルウェールーテルし調印) 会からの助成金によって支えられた: 革新的な領域 (Nos 科学研究費補助金。JP24113514、JP26113710、JP15H05962、および調印、および Nos JP15H05955。JP16H06465、JP16H06464、T.H の JP16K21727)、科研費若手 (B、Nos。JP24770045 と調印の JP26840093)、科研費挑戦的萌芽研究号調印の JP16K14753)。

1 体外 胚珠培養培地の調製 <ol><li>を 体外 の胚珠培養用液体培地 (&#34; N5T 中 &#34;) 1 x Nitsch 基底塩混合物、5% (w/v) トレハロース二水和物、0.05 を含む。% (w/v) の 2-(N morpholino) ethanesulfonic 酸 (MES)-島 (pH 5.8) 1 x Gamborg &#39; s ビタミン ソリューションです</li>。
	<li>島の 5.8 に pH を調整します</li>。
	<li>は、オートクレーブに入れることによって媒体を滅菌 (121 ° C、20 分) または、0.22 μ m フィルターをフィルタ リングします 。
	注: 滅菌媒体格納できる 4 ° C で 2 - 3ヶ月</li>。
</ol> 2。PDMS 駆動式マイクロピラー アレイ素子の作製 <ol><li>カット ナイフ、かみそりの刃または 35 mm ガラス底皿のガラスの部分 (14 mm φ) に収まるようにハサミで PDMS 駆動式マイクロピラー デバイス 。
	注: PDMS 装置建設の完全な手順は前論文 8 , 9 で説明、ここでこうして、詳細は省略します。デバイスがなくても短期的なイメージングのため 4 ウェルまたは 96 ウェルのプレートなどの小さいボリュームでガラス底培養皿を使用できます</li>。
	<li>定置滅菌 15 分の紫外線の下で PDMS 装置の上側</li>
	<li>正方形のチップ ピンセットを使用してによって PDMS 装置を切り、底を殺菌する 15 分の軽い UV の下でそれを維持します</li>。
	<li>35 mm ディッシュに滅菌の PDMS デバイスを転送し、デバイスは培地 (約 5-7 mL) で完全に浸漬されるまで N5T 培地を追加します</li>。
	<li>料理を真空チャンバーに入れて、ガスを抜き圧力を減らします。一夜にしてデバイスの空気が媒体によって置き換えられるまで 3 時間掃除機を維持します 。
	注: デバイスは、強力な真空の下で空気の泡で切り離すことが</li>。
	<li>皿から正方形のチップ ピンセットでそれを保持することによって、デバイスを取るし、サイドと下部に余分なメディアを取り出してペーパー タオルの上に置く 。
	注: これが胚珠の耕作のために使用されるため、駆動式マイクロピラー アレイ (上面) に媒体を削除しないでください</li>。
	<li>アッパー サイドとして駆動式マイクロピラーの部分を保つために確保、76 mm × 26 mm スライド ガラス上にデバイスを転送し、次の胚珠抽出中に完全に乾くことから媒体を防ぐために 35 mm の文化料理ふた付きのデバイスをカバーします</li>。
</ol> 3。Silique 郭清と胚珠抽出 <ol><li>70% エタノールで拭くことによって針 (0.40 mm G) と高級ピンセットを消毒します。針は、注射器や木の棒に接続し、簡単に処理します</li>。
	<li>は、植物の花序をチェックし、実験のための適切な siliques を選択します。約 5 mm siliques 含む受精卵と 8-10 mm siliques 若い球状胚があります</li>。
	<li>は、ピンセットを使用して、siliques を消費税し、76 × 26 mm スライド グラス上に両面テープの上に置きます。について 1 つの silique が含まれています 40 60 胚珠と 3-4 siliques (すなわち 120-240 胚珠) が 1 つのデバイスにとって十分です</li>。
	<li>は、滅菌注射針と顕微鏡下で、ピンセットを使用して胚珠を表示する silique (子房壁) を開きます。胚珠は破損していないように子房壁のみをカットします</li>。
	<li>(2.7 の手順で準備) PDMS のデバイスに N5T 中に開かれた silique を転送し、針やピンセットで媒体に胚珠をリリースします</li>。
	<li>駆動式マイクロピラー アレイ内のスペースに胚珠をプッシュする PDMS デバイス上に小さなカバー ガラス (18 mm × 18 mm) を配置します</li>。
	<li>余分なメディアを削除するピンセットや指を使用して横に引いてカバーガラスを脱ぐ</li>。
	<li>PDMS デバイスを裏返し、35 mm ガラス底皿に置き、少しガラスに固執する正方形のチップ ピンセットを使用してを押してデバイスを押し</li>。
	<li>ガラス底にそっと注ぐ N5T 媒体まで PDMS 装置は中、ずぶ濡れ中瓶をデカントで皿し、パラフィン フィルムが付いている皿をシールします 。
	注: PDMS デバイスは、リサイクルすることができますが、あまりにも古いデバイスは、ガラス底から容易に切り離します。手順 2.5 では完全に脱がないまたは媒体は余りにすぐに注がれる場合デバイスがさらに、浮かぶかもしれない</li>。
</ol> <img alt="Figure 2"class="xfigimg"src="/files/ftp_upload/55975/55975fig2.jpg"/> 図 2: 試料の概略プロシージャ 。
このフロー図は、手順 3.5 から 4.1 に対応します。<a href="//ecsource.jove.com/files/ftp_upload/55975/55975fig2large.jpg"target="_blank"> この図の拡大版を表示するのにはここをクリックしてください</a>。
 4 低速度撮影画像 倒立顕微鏡、3.9 ステップで準備されたガラス底皿を配置し集中する適切な胚珠をオン] <ol><li>. 。
	注: ステージ、位置、および方向で、胚珠が異なりますので適切な胚珠発見されるべき自分のマーカーの蛍光をチェックしています</li>。
	<li>は、メーカーによるとライブ イメージングを開始 &#39; 顕微鏡システムの指示 。
	注: 顕微鏡装置およびパラメーター多様であり従ってユーザーが実験目的に合った適切な設定を選択する必要があります。例として、この原稿で使用される設定は、表 1 に示す</li>。
</ol> <table border="1"fo:keep-together.within-page="1"fo:keep-with-next.within-page="always"> <tbody><tr><td>機器/設定</td> <td>図 3 (A) および補足のビデオ 1 4</td> <td>図 3 (B) および補足のビデオ 2</td> <td>補足動画 3</td></tr> <tr><td>顕微鏡</td> <td>倒立レーザー走査顕微鏡 (A1R MP)</td> <td>回転ディスク共焦点倒立顕微鏡 (CSU W1)</td> <td>ボックス型逆安定したインキュベーション室 (CV1000) と共焦点顕微鏡システム</td></tr> <tr><td>レーザー</td> <td>フェムト秒パルス レーザー</td> <td>488 nm および 561 nm の LD レーザ</td> <td>488 nm および 561 nm の LD レーザ</td></tr> <tr><td>O組込めますレンズ</td> <td>水浸対物レンズ × 40 (NA = 1.15) 浸漬媒体</td> <td>シリコーン油浸対物レンズ X 60 (NA = 1.30)、ピエゾ フォーカス ドライブにマウントされている</td> <td>40 X 対物レンズ (NA = 0.95)</td></tr> <tr><td>検出または</td> <td>外部非 descanned GaAsP PMT 検出器</td> <td>EMCCD カメラ</td> <td>EMCCD カメラ</td></tr> <tr><td>ダイクロイック ミラー</td> <td>DM495 と DM560</td> <td>DM488/561</td> <td>DM400-410/488/561</td></tr> <tr><td>フィルター</td> < td> バンドパス フィルター;534/30 nm と 578/105 nm</td> <td>バンドパス フィルター; 520/35 nm、および 593/46 nm</td> <td>バンドパス フィルター; 520/50 nm と 617/73 nm</td></tr> <tr><td>z 軸に沿ったスライス</td> <td>1 μ m 間隔で 31 z スタック</td> <td>17 z スタック 3 μ m の間ヴァルス</td> <td>5 μ m 間隔で 7 z スタック</td></tr> <tr><td>時間</td> <td>20 分</td> <td>5 分</td> <td>10 分</td></tr></tbody></table> テーブル 1:顕微鏡システム、この原稿で使用される設定します 。
顕微鏡とパラメーターは多様であり従って各ユーザーは、実験のための適切なシステムを選択してください。
<ol start="3"> <li>取得イメージ シーケンスを確認し、.avi またはプレゼンテーションに .mov などの一般的なムービー形式に変換します 。
	注: 顕微鏡のソフトウェアは、ムービー形式として、映像を出力できませんは、ImageJ、NIH イメージ、ファイルの種類を変更するに触発されたオープン ソース プログラムで開いて .tif として画像を保存することが可能。ImageJ を提供しています: https://imagej.nih.gov/ij/。ImageJ は、Z s を作るためにも使用できます。説明したように最大強度投影など、ムービーを挟む: https://imagej.net/Z-functions です</li>。
</ol>

<ol>
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本稿では、単純な体外胚珠栽培プロトコルは、受精卵や胚の開発の住セルイメージ投射で使用するため効率的を紹介します。
PDMS 装置の設計は、胚の段階に応じて最適化を必要があります。開発の最初のデバイスは microcage 配列方向を調整し、胚珠9の位置を修正してより効率的に胚珠をトラップし、駆動式マイクロピラー デバイスを構築しまし?...

単細胞受精卵から有機体の基本的なボディ計画を開発します。ほとんどの顕花植物のココヤシ部門は、樹状突起と撮影やルート、1それぞれに発展する基底のセルを生成します。したがって、胚発生に伴う植物体を形成する方法を理解することが重要だが、彼らは花に深く成長するのでリビング受精および胚のダイナミクスを直接観測する効果的なツールをされていません。トウモロコシ、米などのいくつかの単子葉種体外受精法は確立された2,3をされています。この方法では分離精子と卵細胞を電気的または化学的に、融合し、生成されたセル、肥沃な工場に開発。ただし、双子葉植物植物の雄性と雌性の配偶子の4、5の細胞周期の非同期状態のためおそらく適切な胚を作り出すことができる体外受精法ではありません。さらに、胚周囲組織 (胚乳) は、胚の開発6で重要な役割を果たしています。
モデルの双子葉植物の種、シロイヌナズナ、体外の栽培方法は、胚と胚乳の7を含む全体の胚珠に焦点を当てによって開発されました。このシステムは正常に様々 な化学試薬の胚発生に及ぼすを分析に使用されたが、低い生存率をもっているため時間経過イメージ投射のため適していません。そこで、新規培養胚珠の栽培システムは、受精卵の段階として早期に開始高比8で肥沃な植物を生成するために開発されました。様々 な試験後 Nitsch 培地が分かったし、トレハロースが胚珠8の生存率を大幅に向上します。さらに、胚珠の展開としては成長して、従って頻繁に顕微鏡の観察フィールドから移動、ために、9の中の胚珠を修正する PDMS 装置が開発されました。PDMS デバイスは、心期胚には受精卵から開発をトレースするのに十分である 3-4 日間の長期的なイメージングが有効になります。このメソッドを使用して、それは偏波受精卵・胚のパターンだけでなく通常の条件下でも低分子阻害剤の存在下でまたは様々 な突然変異体の背景8,10 のダイナミクスを可視化することが可能になります。 ,11。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55975/55975fig1.jpg"> 
図 1: 視覚化受精し胚を胚珠を使用特定の蛍光マーカーの模式図。 シロイヌナズナの受精卵は、花の奥深くに生成される胚珠の胚に開発しています。体外培養システム、受精卵・胚胚珠、観察、従って他の胚珠組織で表されない特定の蛍光マーカーを使用することが重要です。 <a href="//ecsource.jove.com/files/ftp_upload/55975/55975fig1large.jpg" target="_blank">この図の拡大版を表示するのにはここをクリックしてください</a>。

<table><tbody><tr><td>Nitsch basal salt mixture</td><td>Duchefa</td><td>N0223</td><td></td></tr><tr><td>trehalose dihydrate</td><td>Wako Pure Chemical</td><td>206-18455</td><td></td></tr><tr><td>MES</td><td>Dojindo</td><td>345-01625</td><td></td></tr><tr><td>Gamborg&amp;rsquo;s vitamin solution</td><td>Sigma-Aldrich</td><td>G1019</td><td></td></tr><tr><td>35-mm glass-bottom dish</td><td>Matsunami Glass</td><td>D111300</td><td></td></tr><tr><td>35 mm culture dish</td><td>Corning</td><td>430588</td><td></td></tr><tr><td>PDMS</td><td>Dow Corning Co.</td><td>Sylgard184</td><td></td></tr><tr><td>76 &amp;times; 26 mm slide glass</td><td>Matsunami Glass</td><td>S1225</td><td></td></tr><tr><td>18 &amp;times; 18 mm slide glass</td><td>Matsunami Glass</td><td>C018181</td><td></td></tr><tr><td>needle (gauge 0.40 mm)</td><td>Terumo</td><td>NN-2719S</td><td></td></tr><tr><td>Immersion medium Immersol W 2010</td><td>Zeiss</td><td>444969-0000-000</td><td></td></tr><tr><td>A1R MP</td><td>Nikon</td><td>A1RsiMP(1080) Ti-E-TIRF</td><td></td></tr><tr><td>CSU-W1</td><td>Yokogawa Electric</td><td></td><td>It is a customized equipment, and thus Catalog Number is not avairable.</td></tr><tr><td>CV1000</td><td>Yokogawa Electric</td><td>CV1000-SP84</td><td></td></tr></tbody></table>

in vitro ovule cultivation for live-cell imaging of zygote polarization and embryo patterning in arabidopsis thaliana

ほとんどの顕花植物の受精および胚は母組織の奥深くに隠されて、従ってそれは長年どのように彼らを動的に開発の謎たとえば、どのように受精は体軸や器官形成の時に胚が様々 な細胞運命を指定する方法を確立する分極します。本稿では、受精とシロイヌナズナの胚の開発の住セルイメージ投射を実行する胚珠体外培養法について説明します。最適化された培地では、受精卵や初期胚肥沃な植物に成長することができます。ポリジメチルシロキサン (PDMS) 駆動式マイクロピラー アレイ デバイスと組み合わせて、胚珠は同じ位置に液体培地で開催されます。この固定は、ココヤシの部門から数日間胚後期に顕微鏡下で同じ胚珠を観察することが重要です。結果住セルイメージ投射を使用して核移行と細胞骨格の再編とも細胞分裂のタイミングや胚パターン形成時の細胞運命仕様など、受精卵の偏波の実時間ダイナミクスを監視できます。さらに、胚発育に及ぼす諸因子の影響を分析する阻害剤治療と細胞間コミュニケーションの役割を調べるレーザー破壊などの光の操作は、この胚珠培養システムを結合できます。

このメソッドはこの胚珠培養システムを用いた偏波受精卵・胚パターン形成生活動態をトレースできます。これは、以前母親組織に深く隠されている受精卵及び胚のリアルタイム挙動を可視化する技術がなかったので成果です。図 3 aと補足のビデオ 1若い受精卵における微小管 (MTs) 穎地域10の横のリングとして蓄?...

Watch this Scientific Journal Video about In Vitro Ovule Cultivation for Live-cell Imaging of Zygote Polarization and Embryo Patterning in Arabidopsis thaliana at JoVE.com

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

本稿では体外胚珠栽培手法についてシロイヌナズナ受精および胚の住セルイメージ投射を可能にします。このメソッドを利用して、受精卵の偏波の中に細胞内動態と初期胚細胞運命決定を可視化します。

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; ...

in-vitro-ovule-cultivation-for-live-cell-imaging-zygote-polarization

Research

JoVE Journal

Developmental Biology

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

10.8K ビュー.  Nagoya University. 本稿では体外胚珠栽培手法についてシロイヌナズナ受精および胚の住セルイメージ投射を可能にします。このメソッドを利用して、受精卵の偏波の中に細胞内動態と初期胚細胞運命決定を可視化します。

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

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

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

胸腺の特性を用いてマウス胚における前駆細胞をセトリング<em&gt;インビボ</em&gt;と<em&gt;インビトロ</em&gt;アッセイ

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

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 ...

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. ...

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

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, ...

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses with an egg cell or a central cell. Thus, two independent fertilization events take place almost simultaneously. The fertilized egg cell and central cell develop into zygote and endosperm, respectively. Therefore, precise control of double fertilization is essential for the ensuing seed development. Double fertilization occurs in the female gametophyte (embryo sac), which is deeply hidden and covered with thick ovule and ovary tissues. This pistil tissue construction makes observation and analysis of double fertilization quite difficult and has created the present situation in which many questions regarding the mechanism of double fertilization remain unanswered. For the functional evaluation of a potential candidate for fertilization regulator, phenotypic analysis of fertilization is important. To judge the completion of fertilization in Arabidopsis thaliana, the shapes of fluorescence signals labeling sperm nuclei are used as indicators. A sperm cell that fails to fertilize is indicated by a condensed fluorescence signal outside of the female gametes, whereas a sperm cell that successfully fertilizes is indicated by a decondensed signal due to karyogamy with the female gametes&#8217; nucleus. The method described here provides a tool to determine successful or failed fertilization under in vivo conditions.

Evaluation of Fertilization State by Tracing Sperm Nuclear Morphology in Arabidopsis Double Fertilization

We demonstrate a method to determine successful or failed fertilization on the basis of sperm nuclear morphology in Arabidopsis double fertilization using an epifluorescence microscope.

アラビドプシス二重受精における精子核形態をたどる受精状態の評価

Flowering plants have a unique sexual reproduction system called &#8216;double fertilization&#39;,&#160;in which each of the sperm cells precisely fuses ...

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 ...

生物学

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 ...

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female gametophyte are essential for double fertilization and subsequent seed development. The interactions between male and female gametophytes during pollen tube reception culminate in pollen tube rupture and the release of two sperm cells to effect double fertilization. As pollen tube growth and double fertilization are deeply hidden within the tissues of the flower, this process is difficult to observe in vivo.
A semi-in vitro (SIV) method for the live-cell imaging of fertilization in the model plant Arabidopsis thaliana has been developed and implemented in several investigations. These studies have helped to elucidate the fundamental features of how the fertilization process occurs in flowering plants and which cellular and molecular changes occur during the interaction of the male and female gametophytes. However, because these live cell imaging experiments involve the excision of individual ovules, they are limited to a low number of observations per imaging session, making this approach tedious and very time-consuming. Among other technical difficulties, a failure of the pollen tubes to fertilize the ovules in vitro is&#160;often reported, which severely confounds such analyses.
Here, a detailed video protocol for the imaging of pollen tube reception and fertilization in an automated and high-throughput manner is provided, allowing for up to 40 observations of pollen tube reception and rupture per imaging session. Coupled with the use of genetically encoded biosensors and marker lines, this method enables the generation of large sample sizes with a reduced time investment. Nuances and critical points of the technique, including flower staging, dissection, medium preparation, and imaging, are clearly detailed in video format to facilitate future research on the dynamics of pollen tube guidance, reception, and double fertilization.

Live Imaging of Arabidopsis Pollen Tube Reception and Double Fertilization Using the Semi-In Vitro Cum Septum Method

Here, we describe an improvement of the semi-in vitro (SIV) method for observing pollen tube guidance and reception in Arabidopsis thaliana,&#160;which increases the receptivity of ovules. The high-throughput SIV cum septum method may be coupled with gametophyte marker lines and genetically encoded biosensors to monitor the dynamic process of fertilization.

セミインビトロ中隔法によるシロイヌナズナの花粉管受容と二重受精のライブイメージング

In flowering plants, the growth and guidance of the pollen tube (male gametophyte) within the pistil and the reception of the pollen tube by the female ...

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

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

胸腺の特性を用いてマウス胚における前駆細胞をセトリング

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

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

シロイヌナズナの胚発生の特性評価法

アラビドプシス二重受精における精子核形態をたどる受精状態の評価

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

セミインビトロ中隔法によるシロイヌナズナの花粉管受容と二重受精のライブイメージング

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

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

胸腺の特性を用いてマウス胚における前駆細胞をセトリング

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

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

シロイヌナズナの胚発生の特性評価法

アラビドプシス二重受精における精子核形態をたどる受精状態の評価

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

セミインビトロ中隔法によるシロイヌナズナの花粉管受容と二重受精のライブイメージング

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