著者は、親切にゼブラフィッシュSOX10プロモーター試薬を共有するためのロバートKelshに感謝します。

1。光変換のための胚コレクション<ol><li>夕方5と6時間楓トランスジェニックゼブラフィッシュ：SOX10の少なくとも10ペアをセットアップします。 </li><li>翌朝、仕切りを引き、正午頃、卵を収集します。ペトリ皿に転送し、28.5℃のインキュベーターに入れます。 </li><li>約24時間後に受精では、死んだ胚を削除してペトリ皿をきれいに。光照射野顕微鏡および強化された緑色蛍光タンパク質（EGFP）フィルターを持つ蛍光顕微鏡を用いて胚13の発達段階を確認してください。楓はこのフィルタを通して見えるようになります。 </li><li>胚は別々のペトリ皿に60 HPF転送ファイブ鮮やかな蛍光の胚に到達し、必要に応じてdechorionateます。 </li></ol> 2。光変換のための胚の取り付け<ol><li>通常の蛍光顕微鏡を用いて胚をマウントします。共焦点顕微鏡で胚をマウントすると、非常に困難である。 </li><li>スタンドを使用してくださいARDのE3水または以下のレシピを使用して、新鮮なE3を準備します。 1X E3Lを作るために以下を追加し、蒸留水のdH 2 Oを1リットルあたり、以下を追加します。 0.292グラム5.0のNaCl 0.013グラム0.17mMのKCl 0.044グラム0.33mmでのCaCl 2（乾燥剤、96％、ACS試薬） 0.081グラム0.33のMgSO 4（無水化） オプション：殺菌剤と攪拌などの1X E3 0.05％メチレンブルー200μlのを追加します。 培地を1週間室温で保持します。 </li><li>標準トリカインを使用するか、以下のレシピを使用してトリカインを準備します。 0.2％の5のLは、以下の成分を混合トリカイン： 45ミリリットルのトリス-Cl（pH9.5で1M） 5 LH 2 O 10グラムのトリカイン</li><li> 46.​​25ミリリットルE3に3.75ミリリットル、0.2％トリカインを追加することで、「映画のジュース」（50ミリリットルE3、0.015％のトリカイン溶液）を準備します。 </li><li> 50mlのガラスフラスコ中で46.35 mgの低融点（LMP）アガロースE3 46.25ミリリットルの水を混合することによって調製し、アガロースマイクロ波処理することにより固体粒子を溶解する。 [追加アガロースおよび使用するまで50℃の水浴中にアガロースを有するガラスフラスコを置くために3.73ミリリットルの0.2％トリカイン。 </li></ol> 3。胚の作製と取り付け<ol><li>チャンバースライドにステップ1.1.2および移植胚で行われたE3/0.015％のトリカイン溶液で胚を麻酔。 </li><li>ティッシュペーパーや胚の上にアガロースプットを持つすべての過剰E3を吸収。 </li><li>位置胚は完全にmicroloaderのヒントと背側。全胚アガロースの上に浮くが、全身を押し下げないようにしてください。チャンバーが完全に流体で満たされるまで、その後、アガロース埋め込ま胚かけE3/0.015％のトリカイン水溶液を注ぐ。 </li></ol> 4。共焦点顕微鏡上でソフトウェアの設定を行い<ol><li>胚を集束させるため、20X空気浸漬可能な目的（ピンホールサイズ20の開口数0.75）を使用します。その後、顕微鏡でL100ライトボタンを押すとソフトウェアチャンネルを選択します。アイコンの表示/取得controls/A1settingsとCLICを開く共焦点セットアップボタンをk個。次の「自動」をクリックして、再度ウィンドウを閉じる前に、以下のチャンネルを選択します。 CH1：DAPIチャネル403.4ナノメートル（350から410 nmの波長の長さを使用することができます） CH2：488.0 nmの CH3：561.9 nmの</li></ol> 5。光変換<ol><li> Ch1のと3の電源をオフにし、[削除]インターロックボタン、スキャンをクリックする前に、Ch2の電源をオンにします。 </li><li>光変換のための目的の細胞に焦点を当てているで1frame/sec、高電圧（HV）200から始まり、その後、フレーム/秒に上がっていくし、それに応じて周囲の1/32フレーム/秒と、HVに到達するまで、HVを下げることによって行わ最高の背景ノイズに応じて100〜120、。 </li><li>胚が合うと、画面の右端に見える緑色の縁をつかんで、それを光変換し、マウスで右クリックを関心領域にフィットするので、それを小さくする。光変換は不可逆的であるので、小さい方が良いです。 </li><li> 200フレーム/秒の1と、HVの電源を入れて、ヒットスキャンします。光変換の領域の画像に拡大表示されるようになりました。 </li><li> CH1（403.4 nm）をオンすることによりPhotoconvert細胞。 GFPチャンネルを通して見た緑の楓がほぼ存在しないまで領域の大きさに応じて、任意の場所に5〜60秒からphotoconvertingレーザにさらされている。 </li><li>緑の楓信号がほぼ存在しない場合、チャネル1をオフにしてください。 CH2および3をオンにします。その後、A1のスキャン領域のフィールド上のウィンドウに右クリックして、目的の細胞をphotoconvertedたかどうかをチェックする「元のサイズに戻る」を押してください。 </li></ol> 6。 zスタックを作る<ol><li>メニューバーのアイコン &#39;キャプチャZシリーズ」を見つけて、それを起動する前に、zスタックの上限と下限を設定してください。 LUTを設定します。 </li></ol> 7。ソフトウェア設定のタイムラプス<ol><li> A1Simple GUIのボックスに移動し、以下の設定をチェック： 1/32フレーム/秒 サイズ1024 一つのループに注意しなければならないZ-スタックの数に応じて、平均4又は8X </li><li>にアクセスしてください。ビュー/買収コントロール/ ND系列の取得および設定した時間枠（8 - 30分）、連続クリックして、zスタックをクリックします。前にテストとして境界線を設定し、ムービーを開始します。 </li><li>終日E3/0.015％のトリカイン溶液チャンバースライドを追加し続ける。夜には、完全にチャンバーを埋める。つまり、次の朝まで続く。 </li><li>翌朝、E3​​/0.015％のトリカイン液を補充し、胚がまだ生きているかどうかを確認します。蛍光の損失は死を示している。終日リフィルとチェックを続ける。 </li><li>構造が形成を終了した、または胚が死亡したときは、映画を仕上げる。 </li></ol> 8。ポストプロダクション処理<ol><li>ツールバーの「ショー最大値投影」をクリックして、右の画像をクリックして、新しいドキュメントを作成します。ビデオは現在、可能な限り最高の品質で表示されます。必要とされていないフレームを削除して、LUTを調整するために行く。最終的には。AVIとして保存します。この形式は、Windows用の罰金になります。 Mac用のソフトウェア·TをダウンロードO。MOVまたは。WMVに変換します。 </li></ol>

<ol>
	<li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevalence+at+Birth+of+Cleft+Lip+With+or+Without+Cleft+Palate:+Data+From+the+International+Perinatal+Database+of+Typical+Oral+Clefts+(IPDTOC).">Prevalence at Birth of Cleft Lip With or Without Cleft Palate: Data From the International Perinatal Database of Typical Oral Clefts (IPDTOC).</a> Cleft Palate Craniofac. J. 48 (1), 66-81 (2011).</li><li>Dixon, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleft+lip+and+palate:+understanding+genetic+and+environmental+influences.">Cleft lip and palate: understanding genetic and environmental influences.</a> Nat. Rev. Genet. 12 (3), 167-178 (2011).</li><li>Mangold, E., Ludwig, K. U., Nothen, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakthroughs+in+the+genetics+of+orofacial+clefting.">Breakthroughs in the genetics of orofacial clefting.</a> Trends Mol. Med. 17 (12), 725-733 (2011).</li><li>Kimmel, C. B., Miller, C. T., Moens, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+and+morphogenesis+of+the+zebrafish+larval+head+skeleton.">Specification and morphogenesis of the zebrafish larval head skeleton.</a> Dev. Biol. 233 (2), 239-257 (2001).</li><li>Dougherty, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Fate+Map+of+First+Pharyngeal+Arch+Structures+in+the+sox10:+kaede+Zebrafish+Transgenic+Model.">Embryonic Fate Map of First Pharyngeal Arch Structures in the sox10: kaede Zebrafish Transgenic Model.</a> J. Craniofac. Surg. 23 (5), 1333-1337 (2012).</li><li>Trainor, P. A., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hox+genes,+neural+crest+cells+and+branchial+arch+patterning.">Hox genes, neural crest cells and branchial arch patterning.</a> Curr. Opin. Cell Biol. 13 (6), 698-705 (2001).</li><li>Schilling, T. F., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segment+and+cell+type+lineage+restrictions+during+pharyngeal+arch+development+in+the+zebrafish+embryo.">Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo.</a> Development. 120 (3), 483-494 (1994).</li><li>Swartz, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+a+palatogenic+gene+program+in+zebrafish.">Examination of a palatogenic gene program in zebrafish.</a> Dev. Dyn. 240 (9), 2204-2220 (2011).</li><li>McCollum, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+toxicity+screening+in+zebrafish.+Birth+Defects+Res.">Developmental toxicity screening in zebrafish. Birth Defects Res.</a> C. Embryo Today. 93 (2), 67-114 (2011).</li><li>Wada, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132 (17), 3977-3988 (2005).</li><li>Eberhart, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Hedgehog+signaling+from+neural+to+oral+epithelium+organizes+anterior+craniofacial+development.">Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development.</a> Development. 133 (6), 1069-1077 (2006).</li><li>Ando, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc. Natl. Acad. Sci. U S A. 99 (20), 12651-12656 (2002).</li><li>Kimmel, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203 (3), 253-310 (1993).</li><li>Kawakami, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish-secreted+matrix+protein+you/scube2+is+implicated+in+long-range+regulation+of+hedgehog+signaling.">The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling.</a> Curr. Biol. 15 (5), 480-488 (2005).</li><li>Lombardo, V. A., Sporbert, A., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+tracking+using+photoconvertible+proteins+during+zebrafish+development.">Cell tracking using photoconvertible proteins during zebrafish development.</a> J. Vis. Exp. (67), e4350 (2012).</li></ol>

著者は、彼らが競合する経済的利益を持っていないことを宣言します。

ここゼブラフィッシュモデルにおける頭蓋顔面の開発の可視化のための新たな方法が示されている。 SOX10：楓トランスジェニックゼブラフィッシュ線が正常に詳細にCNCCの移動パターンを記述するために使用されてきたが、モデル生物5として使用される。 以前の研究では、細胞を標的とする眼などのグロスランドマークを使用していると楓mRNAの注入、光変換アッ...

口腔顔面裂が1/700-1で、最も一般的な頭蓋顔面奇形を表し、000配達は1に影響を与えた。初期の発生学的頭蓋顔面の開発の破壊は、口唇口蓋裂（CL / P）の形成につながることができます。症候裂の原因は、主に示してきたが、口腔顔面分裂文形成の非症候性形態の遺伝的およびエピジェネティックな拠点は、まだ2-4を発見する必要があります。これらの奇形の病因および発症機序を理解するためには、セルラーベースで頭蓋顔面構造の発達を解明する必要がある。 すべての脊椎動物種に頭蓋神経堤細胞（CNCC）口腔顔面構造の形成に寄与する咽頭のアーチを、設定するために、背側神経管から移行。初期の発生学的神経堤の開発の破壊は、CL / P 5-7を含む頭蓋顔面奇形の形成につながることができます。 ADでゼブラフィッシュおよび哺乳動物の頭蓋顔面の開発（CNCCsは相同領域に存在する）との間の構造的類似性ditionは、遺伝子制御ネットワークが高度に保存されている。またCNCCsは、ゼブラフィッシュのCL / Pの発生および遺伝的基礎を研究するための強力な生物を作り、羊膜種およびゼブラフィッシュ8の間に同じ様式で発症することが示されているそれは、小型、高速かつ元子宮胚発生、高い繁殖率を含む多くの利点を持っています。さらに、胚を顕微鏡9の下に複雑な発生事象を観察することが影響を受けやすいこと、光学的に透明である。これは、頭蓋神経堤細胞の遊走および分化の研究のための理想的な動物モデルである。 楓トランスジェニックモデル5：以前に公開され仕事8、10、11に拡大して、CNCCの遊走パターンは、SOX10を用いて詳細に説明した。楓はTUフォト転換タンパク質である光活性化後の緑色から赤色へのRNSかつ正確CNCCsをトレースすることができる。この変換の間、ペプチド骨格は、細胞がそれらの最終目的地12まで追跡することができることを意味し、変換が安定であることを示唆して切断される。 SOX10の転写制御下に楓で標識したトランスジェニック系統は、羊膜の口蓋とゼブラフィッシュの篩骨板は前頭鼻隆起（FNP）であり、Y字型融合縫い目の間に類似であることを、二国間上顎プロミネンス（MXP）の融合によって相同的に形成されることを示した種。 他の用途の中でも、SOX10：楓トランスジェニックゼブラフィッシュモデルは、正常および異常な頭蓋顔面構造の形成を示すために、異なる発達段階でのゼブラフィッシュ胚のビデオを生成するために使用された。細胞の特定の基の光変換は、それらの発達を追跡することが可能となる。 craniofacを開発するライブイメージングを作成するには、この方法でアプローチしてゼブラフィッシュにおけるIAL構造は、それが簡単に視覚的に、この複雑な発達過程を実証すること、導入される。 例として楓トランスジェニックゼブラ：このプロトコルはSOX10内篩骨プレートの正常な発達を使用してこれらのビデオを生成する経験を共有することを目的としている。このプロトコルは、さらに、ゼブラフィッシュにおける頭蓋神経堤細胞に由来する任意の構造の経時ビデオを作るにも適用することができる。

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>sox10: kaede transgenic zebrafish line</td>
			<td>MGH</td>
			<td></td>
			<td>Available via the Liao lab</td>
		</tr>
		<tr>
			<td>Petri dishes 100x15 mm</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 mmx10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Low melting point (LMP) Agarose</td>
			<td>Invitrogen</td>
			<td>15517022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Tek 2 Chamber SlideSystem</td>
			<td>LabTek</td>
			<td>154453</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloaders 200/pk</td>
			<td>Fisher</td>
			<td>E5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R Si Confocal Ti series</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements Software AR3.2 64-bit</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy

脊椎動物palatogenesisは頭蓋神経堤（CNC）細胞、収束と顔のプロミネンスの延長、および頭蓋顔面骨格の成熟の移行を伴う非常に振り付けで複雑な発達過程です。楓トランスジェニックゼブラフィッシュラインが生成されました：ゼブラフィッシュ口蓋の特定の領域SOX10に頭蓋神経堤の寄与を研究すること。 SOX10は、それによって、伝統的な染料やレポーターmRNAの注入よりもより正確なプロセスを標識し、細胞を作り、神経堤に楓のレポータータンパク質の系統限定を提供しています。楓、光活性化の後に赤色から緑色に変わり、正確に細胞を追跡することができる光転換タンパク質である。 SOX10：楓トランスジェニック系統は、下顎要素対上顎を生じさせると、羊膜に顔面プロミネンスの相同性を示してCNC細胞集団を線引きする系統分析を実行するために使用した。このプロトコルは、手順を説明します楓ゼブラフィッシュ胚：SOX10のライブの時間経過のビデオを生成するために、S。篩骨プレートの開発は、実用的な例となる。このプロトコルは、トランスジェニックゼブラフィッシュのどの楓または類似photoconvertibleレポータータンパク質のタイムラプス共焦点記録を作るに適用することができます。さらに、ゼブラフィッシュ変異体における頭蓋顔面構造のみならず、正常でなく、発達異常を捕捉するために使用することができる。

SOX10に：楓トランスジェニック系統、渡り鳥とポスト渡り鳥CNCCsは蛍光緑のラベルが付いています。緑色蛍光で標識された楓CNCC細胞は、内因性のSOX10 mRNA発現を再現5。 他の用途の中でも、この動物モデルは、より良好なCNCC依存性頭蓋顔面構造の発達を可視化した。特定の構造の正常な発達にも頭蓋顔面奇形の病理学的な開発、特に口唇口蓋裂は、捕獲さ?...

Watch this Scientific Journal Video about Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy at JoVE.com

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

実験データの可視化は、科学界に結果を提示する重要な要素となっています。成長した胚のライブタイムラプス撮影の生成は複雑な発達過程をより良くプレゼンテーションや理解に貢献しています。このプロトコルは、ゼブラフィッシュで楓タンパク質の光変換を経由して細胞標識へのステップバイステップガイドです。

SOX10中の顔面の開発の可視化：楓トランスジェニックゼブラフィッシュラインタイムラプス共焦点顕微鏡を用いて

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

visualization-craniofacial-development-sox10-kaede-transgenic

Research

JoVE Journal

Biology

12.7K ビュー.  Massachusetts General Hospital. 実験データの可視化は、科学界に結果を提示する重要な要素となっています。成長した胚のライブタイムラプス撮影の生成は複雑な発達過程をより良くプレゼンテーションや理解に貢献しています。このプロトコルは、ゼブラフィッシュで楓タンパク質の光変換を経由して細胞標識へのステップバイステップガイドです。 

ビデオ: Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Monitoring Actin Disassembly with Time-lapse Microscopy

タイムラプス顕微鏡で分解アクチンの監視

生物学

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

ライブゼブラフィッシュの胚における二光子軸索切断とタイムラプス共焦点イメージング

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

共焦点顕微鏡によるゼブラフィッシュ胚の脳のイメージングを生きる

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

siRNAトランスフェクション後の有糸分裂のタイムラプスイメージング

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

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 ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

の初期発生のタイムラプス顕微鏡線虫（Caenorhabditis elegans）

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

顔面間葉のエレクトロポレーション

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

タイムラプスビデオ顕微鏡を用いた細胞のランダムな移行の定量分析

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

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 ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

4D共焦点顕微鏡を用いたゼブラフィッシュにおける顔面形態形成を分析する

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...