この作業はtheNIH grantNS042626によってサポートされていました。我々は、クルトソーンとイメージングの支援のためのUCSFニコンイメージングセンターに感謝します。

1。ゼブラフィッシュ胚の調製<ol><li>エレクトロ二日前、女性とは別の男性にディバイダを使用して、野生型の魚の交尾ケージを設定。 </li><li>エレクトロポレーションの前にある日は、前日からケージを交配することで仕切りを引き出します。 </li><li>胚を収集し、28.5で0.003％フェニルチオ尿素（PTU）を含む30ミリリットル胚培地℃で50受精胚をインキュベート</li><li>エレクトロポレーションの日に、胚が22hpfの発達段階に達したときに微細な鉗子（イノックス5、デュモン電子、スイス）と手動で胚dechorionate。その後、0.003％のPTUを含む10ミリリットルエレクトロポリンガーの3（180 mMのNaCl、5mMのKClを、1.8 mMの塩化カルシウム、5mMのHepes緩衝液pH7.2）に胚を移す。 </li><li>呼出音が胚を麻酔だ10mlのエレクトロポレーション、420μlのtricaine株式（4mg/ml）を追加します。 </li></ol> 2。エレクトロポレーションのための準備<ol><li>ガラスの注射針は、フレーミング、ブラウンP897プラー（サッタインスツルメンツ、ノヴァト、カリフォルニア州、米国）で（1.2 mm外径、0.9mmのID、フィラメントを持つ）の毛細血管から引っ張られた。針の先端は、図1Dに示すように鋭い端を得るために鉗子で中断してしまった。先端の直径は10μm程度である必要があり、テーパー角度は30度前後となるはずです。 </li><li>エレクトロポレーションリンゲル溶液で0.8％、1％低融点アガロース（シェルトンサイエンティフィック社カタログ＃IB70050を）準​​備する。 2 mlのマイクロチューブに分注しアガロース溶液。 〜37℃でヒートブロックに分注しておく</li><li> Gal4/UASシステムは、エレクトロポレーション胚の神経前駆細胞における遺伝子発現を駆動するために使用されます。二つのプラスミドが使用されています：プラスミドGal4FFがEF1αユビキタスプロモーターによって駆動されるEF1α- GFF - PT2KXIG、、そしてプラスミドUAS - E1B - EGFP - PT2KXIGを。 </li><li> EF1α- GFF - PT2KXIGとGenEluteTM HPプラスミドミディプレップキット（Sigma）を使用してUAS - E1B - EGFP - PT2KXIGプラスミドDNAを準備します。 </li><li>プラスミドを、10mMトリス· Clの1〜2μgの/μlの最終濃度（PH 8.5）に酢酸ナトリウム/エタノール沈殿により濃縮されています。 </li><li>二つのプラスミドを混合し、各プラスミドに500 ng /μLの中の最終濃度にヌクレアーゼフリー水で希釈する。ソリューションへのフェノールレッドの加算は、（総容積1 / 10に希釈）配信の可視化に適しています。氷の上にDNA溶液を保管してください。 </li><li>右2μlのDNA溶液とエレクトロポレーション、積み込む注射針の前に、2 NLへの注入量を調整する。 </li></ol> 3。エレクトロポレーション<ol><li> steroの解剖顕微鏡（50倍の最大倍率を持つツァイスSTEMI 2000）、エア圧力インジェクター（ナリシゲIM 300インジェクター）、2つのマニピュレーター（WPI M3301R、世界の精密機器、サラソタ、フロリダ州、米国）、及び電気刺激（SD9図1Aに示すように方形パルス刺激、グラスTlefactor、ウエストワーウィ​​ック、ロードアイランド州、米国）が設定されています。 </li><li> 1％低融点アガロースで胚、浸漬2胚をマウントする。その後すぐにガラスピペットでアガロースから胚を削除します。 </li><li>アガロースの個々の滴で反転プラスチックシャーレの蓋に胚を置きます。アガロースは、脳が電極とマイクロインジェクションの針（図1B）の両方にアクセスできるように固化する前に、背アップ位置までファイバープローブで軽く胚を親しませる。胚は、個々のアガロースドロップにマウントされている必要があります。 </li><li>左のマニピュレータで電極を操作する。図1Bに示すように、アガロースに電極を挿入します。カスタムプラチナイリジウムパラレルバイポーラ電極を直径と間隔が500μmで125μmのは、離れてエレクトロポレーション（FHC、カタログ＃PBSA0575）に使用されます。 </li><li>前脳の脳室内にマイクロインジェクションの針を挿入し、赤い液体が流れると心室の腫脹が観察できるように、4nl DNAミックスを注入する。 </li><li>すぐに、一28.5 Vパルス持続的な1ミリ秒は手動で適用した。その後、極性を逆にし、複数28.5 Vパルス持続する1ミリ秒を適用します。これは、前脳の神経前駆細胞の二国間のモザイクのラベルになります。 </li><li> 10胚のエレクトロポレーション後、胚を解放するために顕微ナイフで胚とアガロース皮をカバーするために5ミリリットル胚培地を加える。 </li><li> 28.5で0.003パーセントPTUとインキュベートを含む30ミリリットル胚培地の新鮮な料理℃にエレクトロポレーション胚を転送する</li></ol> 4。マウントとコマ撮りライブイメージング<ol><li>落射蛍光解剖顕微鏡下で観察した場合、エレクトロポレーション後4時間程度、EGFPのシグナルが観測される。 </li><li>エレクトロポレーションから5時間後の、非常にモザイクが前脳の領域に強いEGFPの発現と胚を選択する。 </li><li> 0.003％のPTUを含む10ミリリットル胚培地に選択された胚を移す。 </li><li> 10ミリリットル胚培地tに420μlのtricaine株式（4mg/ml）を追加Oは、胚を麻酔。 </li><li>胚をマウントするには、0.8％低融点アガロースに1胚を浸す。その後すぐにガラスピペットでアガロースから胚を削除と35mmガラスボトムディッシュ（マテック株式会社、部品番号P35GC - 1.5 - 10 - C、の中心に胚を置く<a href="http://www.glass-bottom-dishes.com/">www.glass -ボトムdishes.com</a> ） 。 </li><li>アガロースが固まる前に背アップ位置までファイバープローブで軽く胚を親しませる。 </li><li>図1C（我々は上右の目標とニコンC1スペクトル共焦点顕微鏡を使用）に示すように共焦点顕微鏡の温度制御されたステージ上で適切に皿を置きます。 28.5温度を調整して℃に</li><li> 3ミリリットル28.5 °の胚をカバーするためにPTU 0.003％含むC予熱胚培地を追加。胚は、現在のイメージングのための準備ができています。 </li><li>水浸対物レンズを用いて一定の間隔でタイムラプスライブイメージングを実行します。 </li></ol> 5。代表的な結果図2に示すように22 HPFでEF1α- GFF + UAS - E1B - EGFPのエレクトロ胚のタイムラプス共焦点ライブイメージングのフレームが選択されています。 <img src="/files/ftp_upload/2594/2594fig1.jpg" alt="図1" /> 図1の装置において、設定する。エレクトロポレーション装置の（A）組立：グラスSD9方形パルス刺激（i）を、左のマニピュレータ（II）、電極（III）、ツァイス解剖顕微鏡（IV）、注射針（V）、右のマニピュレータ（VI）とナリシゲIM 300マイクロインジェクター（VII）。 （B）の電極（i）は、前脳脳室（II）及び注射針（iii）の相対位置。赤い色のDNA溶液が前脳の脳室に注入されています注意してください。 （C）ニコンC1共焦点顕微鏡でライブイメージングのアセンブリ：温度調節器（i）は、客観LEN（II）とガラスの底の培養皿（ⅲ）（Dの低融点アガロースに埋め込まれているエレクトロポレーション胚を浸漬する水DNAのマイクロインジェクションのためのプルアップとカット針の）イメージ。 <img src="/files/ftp_upload/2594/2594fig2.jpg" alt="図2" /> 図2。22 HPFでEF1α- GFF + UAS - E1B - EGFPとエレクトロ胚の18時間のタイムラプス共焦点ライブイメージングの選択されたフレーム。心室の表面には、各画像フレームの底面側にあります。スケールバーは10μmを表す。

<ol>
	<li>Temple, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+neural+stem+cells.">The development of neural stem cells.</a> Nature. 414, 112-117 (2001).</li><li>G#246;tz, M., Huttner, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cell+biology+of+neurogenesis.">The cell biology of neurogenesis.</a> Nat. Rev. Mol. Cell Biol. 6, 777-788 (2005).</li><li>Cerda, G. A., Thomas, J. E., Allende, M. L., Karlstrom, R. O., Palma, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+DNA,+RNA,+and+morpholinos+into+zebrafish+embryos.">Electroporation of DNA, RNA, and morpholinos into zebrafish embryos.</a> Methods. 39, 207-211 (2006).</li><li>Hendricks, M., Jesuthasan, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation-based+methods+for+in+vivo,+whole+mount+and+primary+culture+analysis+of+zebrafish+brain+development.">Electroporation-based methods for in vivo, whole mount and primary culture analysis of zebrafish brain development.</a> Neural. Dev. 2, 6-6 (2007).</li><li>Tawk, M., Bianco, I. H., Clarke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Focal+electroporation+in+zebrafish+embryos+and+larvae.">Focal electroporation in zebrafish embryos and larvae.</a> Methods Mol. Biol. 546, 145-151 (2009).</li><li>Tawk, M., Tuil, D., Torrente, Y., Vriz, S., Paulin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-efficiency+gene+transfer+into+adult+fish:+a+new+tool+to+study+fin+regeneration.">High-efficiency gene transfer into adult fish: a new tool to study fin regeneration.</a> Genesis. 32, 27-31 (2002).</li></ol>

利害の衝突は宣言されません。

このビデオでは、我々は開発ゼブラフィッシュの脳のクローンレベルでの神経前駆細胞のタイムラプスライブイメージングのための方法を示しています。我々はテストされ、既存のエレクトロポレーションプロトコル4-6遺伝的および蛍光個々の神経前駆細胞を標識するように変更。少数の細胞がまばらにエレクトロポレーションの相対的な低電圧で標識されて以来、クローン的に関連する子孫細胞で構成される系統樹を確立することができます。 EGFPに加えて、神経前駆細胞は、subcellularly単に他の蛍光マーカーでEGFPを置き換えることにより、種々の蛍光タンパク質で標識することができます。核が赤色蛍光タンパク質で標識される一方で例えば、EF1α- GFFがUAS - E1B - memberane - EGFPとUAS - E1B - H2B - MRFPと共同エレクトロされて​​いる場合、個々の前駆細胞の膜は、緑色蛍光タンパク質で標識されます。ライブイメージング中に観察されたエレクトロ胚といくつかのアポトーシスの一般的な正常な発達により明らかなように標識した前駆細胞のほとんどは健康です。また、胚をマウントし、細胞の健康を保つために共焦点ライブイメージング中に最小限のレーザパワーを使用するためにアガロースの低濃度を使用することが重要です。本技術は、後肢の脳や脊髄などの中枢神経系の他の部分にも適用できます。 DNA構築物をエレクトロポレーションのための心室に注入しなけ​​ればならないしかし、この手法は、18 HPF未満の胚に適用することはできません。

time-lapse live imaging of clonally related neural progenitor cells in the developing zebrafish forebrain

神経前駆細胞の分裂、遊走や分化の正確なパターンは、適切な脳の発達と機能の1,2に不可欠です。 生体内環境における複雑な神経前駆細胞の挙動を理解するために、無傷の脳内の神経前駆細胞のタイムラプスライブイメージングは、批判的に必要となります。このビデオでは、我々はin vivoでの脳の神経前駆細胞の発達を視覚化するためにゼブラフィッシュ胚のユニークな機能を利用する。我々は遺伝的にとまばらに個々の神経前駆細胞を標識するためにエレクトロポレーションを使用してください。簡単に言えば、DNAは、蛍光マーカーのためのコーディング構造22時間後に受精の前脳の脳室に注入した（HPF）ゼブラフィッシュの胚と電気パルスは即座に配信されていました。 6時間後、エレクトロポレーションゼブラフィッシュの胚をガラス底の培養皿に低融点アガロースでマウントされた。蛍光標識した神経前駆細胞は、水浸対物レンズを用いて共焦点顕微鏡下で一定の間隔で36hoursのために画像化した。本方法は、前脳の神経前駆細胞のin vivoでの開発に洞察を得るために手段を提供し、ゼブラフィッシュの胚の中枢神経系の他の部分に適用することができます。

Watch this Scientific Journal Video about Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain at JoVE.com

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

現在のビデオは、開発ゼブラフィッシュの脳内の個々の神経前駆細胞のライブイメージングを実行するためのエレクトロポレーション、共焦点顕微鏡の組み合わせを活用する方法を示しています。<em&gt; in vivoで</emクローンレベルで前脳の神経前駆細胞の発達の&gt;分析は、この方法で達成することができます。

開発ゼブラフィッシュ前脳におけるクローン関連の神経前駆細胞のタイムラプスライブイメージング

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

time-lapse-live-imaging-clonally-related-neural-progenitor-cells

Nanyang Technological University

Research

JoVE Journal

Neuroscience

10.2K ビュー.  University of California San Francisco. 現在のビデオは、開発ゼブラフィッシュの脳内の個々の神経前駆細胞のライブイメージングを実行するためのエレクトロポレーション、共焦点顕微鏡の組み合わせを活用する方法を示しています。 in vivoで分析は、この方法で達成することができます。

ビデオ: Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

無傷の感覚器官の前駆細胞の生細胞イメージングショウジョウバエ蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

神経科学

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

開発神​​経上皮における細胞挙動の高解像度のライブイメージング

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Time-lapse Imaging of Neuroblast Migration in Acute Slices of the Adult Mouse Forebrain

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells in restricted areas of the adult mammalian brain. Newborn neuroblasts from the subventricular zone (SVZ) migrate along the rostral migratory stream (RMS) to their final destination in the olfactory bulb (OB)1. In the RMS, neuroblasts migrate tangentially in chains ensheathed by astrocytic processes2,3 using blood vessels as a structural support and a source of molecular factors required for migration4,5. In the OB, neuroblasts detach from the chains and migrate radially into the different bulbar layers where they differentiate into interneurons and integrate into the existing network1, 6.
In this manuscript we describe the procedure for monitoring cell migration in acute slices of the rodent brain. The use of acute slices allows the assessment of cell migration in the microenvironment that closely resembling to in vivo conditions and in brain regions that are difficult to access for in vivo imaging. In addition, it avoids long culturing condition as in the case of organotypic and cell cultures that may eventually alter the migration properties of the cells. Neuronal precursors in acute slices can be visualized using DIC optics or fluorescent proteins. Viral labeling of neuronal precursors in the SVZ, grafting neuroblasts from reporter mice into the SVZ of wild-type mice, and using transgenic mice that express fluorescent protein in neuroblasts are all suitable methods for visualizing neuroblasts and following their migration. The later method, however, does not allow individual cells to be tracked for long periods of time because of the high density of labeled cells. We used a wide-field fluorescent upright microscope equipped with a CCD camera to achieve a relatively rapid acquisition interval (one image every 15 or 30 sec) to reliably identify the stationary and migratory phases. A precise identification of the duration of the stationary and migratory phases is crucial for the unambiguous interpretation of results. We also performed multiple z-step acquisitions to monitor neuroblasts migration in 3D. Wide-field fluorescent imaging has been used extensively to visualize neuronal migration7-10. Here, we describe detailed protocol for labeling neuroblasts, performing real-time video-imaging of neuroblast migration in acute slices of the adult mouse forebrain, and analyzing cell migration. While the described protocol exemplified the migration of neuroblasts in the adult RMS, it can also be used to follow cell migration in embryonic and early postnatal brains.

We describe a protocol for real-time videoimaging of neuronal migration in the mouse forebrain. The migration of virally-labeled or grafted neuronal precursors was recorded in acute live slices using wide-field fluorescent imaging with a relatively rapid acquisition interval to study the different phases of cell migration, including the durations of the stationary and migration phases and the speed of migration.

成体マウスの前脳の急性スライスにおける神経芽細胞の移行のタイムラプスイメージング

There is a substantial body of evidence indicating that new functional neurons are constitutively generated from an endogenous pool of neural stem cells ...

Forebrain Electrophysiological Recording in Larval Zebrafish

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked seizures, epilepsy can be acquired as a result of an insult to the brain or a genetic mutation. Efforts to model seizures in animals have primarily utilized acquired insults (convulsant drugs, stimulation or brain injury) and genetic manipulations (antisense knockdown, homologous recombination or transgenesis) in rodents. Zebrafish are a vertebrate model system1-3 that could provide a valuable alternative to rodent-based epilepsy research. Zebrafish are used extensively in the study of vertebrate genetics or development, exhibit a high degree of genetic similarity to mammals and express homologs for ~85% of known human single-gene epilepsy mutations. Because of their small size (4-6 mm in length), zebrafish larvae can be maintained in fluid volumes as low as 100 &mu;l during early development and arrayed in multi-well plates. Reagents can be added directly to the solution in which embryos develop, simplifying drug administration and enabling rapid in vivo screening of test compounds4. Synthetic oligonucleotides (morpholinos), mutagenesis, zinc finger nuclease and transgenic approaches can be used to rapidly generate gene knockdown or mutation in zebrafish5-7. These properties afford zebrafish studies an unprecedented statistical power analysis advantage over rodents in the study of neurological disorders such as epilepsy. Because the "gold standard" for epilepsy research is to monitor and analyze the abnormal electrical discharges that originate in a central brain structure (i.e., seizures), a method to efficiently record brain activity in larval zebrafish is described here. This method is an adaptation of conventional extracellular recording techniques and allows for stable long-term monitoring of brain activity in intact zebrafish larvae. Sample recordings are shown for acute seizures induced by bath application of convulsant drugs and spontaneous seizures recorded in a genetically modified fish.

A simple method to record extracellular field potentials in the larval zebrafish forebrain is described. The method provides a robust in vivo read-out of seizure-like activity. This technique can be used with genetically modified zebrafish larvae carrying epilepsy-related genes or seizures evoked by administration of convulsant drugs.

ゼブラフィッシュ幼生における前脳電気生理学的記録

Epilepsy affects nearly 3 million people in the United States and up to 50 million people worldwide. Defined as the occurrence of spontaneous unprovoked ...

Motor Nerve Transection and Time-lapse Imaging of Glial Cell Behaviors in Live Zebrafish

The nervous system is often described as a hard-wired component of the body even though it is a considerably fluid organ system that reacts to external stimuli in a consistent, stereotyped manner, while maintaining incredible flexibility and plasticity. Unlike the central nervous system (CNS), the peripheral nervous system (PNS) is capable of significant repair, but we have only just begun to understand the cellular and molecular mechanisms that govern this phenomenon. Using zebrafish as a model system, we have the unprecedented opportunity to couple regenerative studies with in vivo imaging and genetic manipulation. Peripheral nerves are composed of axons surrounded by layers of glia and connective tissue. Axons are ensheathed by myelinating or non-myelinating Schwann cells, which are in turn wrapped into a fascicle by a cellular sheath called the perineurium. Following an injury, adult peripheral nerves have the remarkable capacity to remove damaged axonal debris and re-innervate targets. To investigate the roles of all peripheral glia in PNS regeneration, we describe here an axon transection assay that uses a commercially available nitrogen-pumped dye laser to axotomize motor nerves in live transgenic zebrafish. We further describe the methods to couple these experiments to time-lapse imaging of injured and control nerves. This experimental paradigm can be used to not only assess the role that glia play in nerve regeneration, but can also be the platform for elucidating the molecular mechanisms that govern nervous system repair.

Although the peripheral nervous system (PNS) is capable of significant repair after injury, little is known about the cellular and molecular mechanisms that govern this phenomenon. Using live, transgenic zebrafish and a reproducible nerve transection assay, we can study dynamic glial cell behaviors during nerve degeneration and regeneration.

モーター神経切断とライブゼブラフィッシュにおけるグリア細胞の挙動のタイムラプスイメージング

The  nervous system is often described as a hard-wired component of the body even  though it is a considerably fluid organ system that reacts to external ...

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to neuroblasts able to move along the rostral migratory stream (RMS) towards the olfactory bulb (OB). This long-distance migration is required for the subsequent maturation of newborn neurons in the OB, but the molecular mechanisms regulating this process are still unclear. Investigating the signaling pathways controlling neuroblast motility may not only help understand a fundamental step in neurogenesis, but also have therapeutic regenerative potential, given the ability of these neuroblasts to target brain sites affected by injury, stroke, or degeneration.
In this manuscript we describe a detailed protocol for in vivo postnatal electroporation and subsequent time-lapse imaging of neuroblast migration in the mouse RMS. Postnatal electroporation can efficiently transfect SVZ progenitor cells, which in turn generate neuroblasts migrating along the RMS. Using confocal spinning disk time-lapse microscopy on acute brain slice cultures, neuroblast migration can be monitored in an environment closely resembling the in vivo condition. Moreover, neuroblast motility can be tracked and quantitatively analyzed. As an example, we describe how to use in vivo postnatal electroporation of a GFP-expressing plasmid to label and visualize neuroblasts migrating along the RMS. Electroporation of shRNA or CRE recombinase-expressing plasmids in conditional knockout mice employing the LoxP system can also be used to target genes of interest. Pharmacological manipulation of acute brain slice cultures can be performed to investigate the role of different signaling molecules in neuroblast migration. By coupling in vivo electroporation with time-lapse imaging, we hope to understand the molecular mechanisms controlling neuroblast motility and contribute to the development of novel approaches to promote brain repair.

In vivo Postnatal Electroporation and Time-lapse Imaging of Neuroblast Migration in Mouse Acute Brain Slices

Neuroblast migration is a fundamental event in postnatal neurogenesis. We describe a protocol for efficient labeling of neuroblasts by in vivo postnatal electroporation and subsequent visualization of their migration using time-lapse imaging of acute brain slices. We include a description for the quantitative analysis of neuroblast dynamics by video tracking.

生体産後エレクトロポレーションおよびマウスの急性脳スライスにおける神経芽細胞移動のタイムラプスイメージング中

The subventricular zone (SVZ) is one of the main neurogenic niches in the postnatal brain. Here, neural progenitors proliferate and give rise to ...

Assessing Cell Cycle Progression of Neural Stem and Progenitor Cells in the Mouse Developing Brain after Genotoxic Stress

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a pseudostratified epithelium lining the lateral ventricles of the embryonic brain. Genotoxic stresses, such as ionizing radiation, have highly deleterious effects on the developing brain related to the high sensitivity of NSPC. Elucidation of the cellular and molecular mechanisms involved depends on the characterization of the DNA damage response of these particular types of cells, which requires an accurate method to determine NSPC progression through the cell cycle in the damaged tissue. Here is shown a method based on successive intraperitoneal injections of EdU and BrdU in pregnant mice and further detection of these two thymidine analogues in coronal sections of the embryonic brain. EdU and BrdU are both incorporated in DNA of replicating cells during S phase and are detected by two different techniques (azide or a specific antibody, respectively), which facilitate their simultaneous detection. EdU and BrdU staining are then determined for each NSPC nucleus in function of its distance from the ventricular margin in a standard region of the dorsal telencephalon. Thus this dual labeling technique allows distinguishing cells that progressed through the cell cycle from those that have activated a cell cycle checkpoint leading to cell cycle arrest in response to DNA damage.
An example of experiment is presented, in which EdU was injected before irradiation and BrdU immediately after and analyzes performed within the 4 hr&#160;following irradiation. This protocol provides an accurate analysis of the acute DNA damage response of NSPC in function of the phase of the cell cycle at which they have been irradiated. This method is easily transposable to many other systems in order to determine the impact of a particular treatment on cell cycle progression in living tissues.

Administration of two analogs of thymidine, EdU and BrdU, in pregnant mice allows the analysis of cell cycle progression in neural and progenitor cells in the embryonic mouse brain. This method is useful to determine the effects of genotoxic stress, including ionizing radiation, during brain development.

神経幹細胞の細胞周期の進行を評価し、マウスでの前駆細胞は、遺伝毒性ストレス後の脳の開発

Neurons of the cerebral cortex are generated during brain development from different types of neural stem and progenitor cells (NSPC), which form a ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

発生中のマウス胚性皮質における有糸分裂のライブイメージング

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

ヒト多能性幹細胞からの神経堤前駆細胞のフィーダーフリー導出

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building the highly ordered, complex structure of the mammalian cochlea proceeds for around ten days. In order to study changes in gene expression over this period and their response to pharmaceutical or genetic manipulation, long-term imaging is necessary. Previously, live imaging has typically been limited by the viability of explanted tissue in a humidified chamber atop a standard microscope. Difficulty in maintaining optimal conditions for culture growth with regard to humidity and temperature has placed limits on the length of imaging experiments. A microscope integrated into a modified tissue culture incubator provides an excellent environment for long term-live imaging. In this method we demonstrate how to establish embryonic mouse cochlear explants and how to use an incubator microscope to conduct time lapse imaging using both bright field and fluorescent microscopy to examine the behavior of a typical embryonic day (E) 13 cochlear explant and Sox2, a marker of the prosensory cells of the cochlea, over 5 days.

Live imaging of the embryonic mammalian cochlea is challenging because the developmental processes at hand operate on a temporal gradient over ten days. Here we present a method for culturing and then imaging embryonic cochlear explant tissue taken from a fluorescent reporter mouse over five days.

マウス蝸牛外植片の長期的タイムラプスイメージング

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...