私たちは、魚のケアのためのCチャンに感謝します。この作品は、中国の国家自然科学基金（GPへ31171074、31371099と31571067）によって及び浦江タレントプロジェクト（GPへ09PJ1401900）によってサポートされていました。

ゼブラフィッシュ実験は、復旦大学施設内動物管理使用委員会によって承認されました。ゼブラフィッシュは、標準的な手順20に従って上昇させて飼育しました。 1.準備<ol><li>幼虫のゼブラフィッシュDPF 17 DPFと25を育成<ol><li>交差前日午後遅く交差タンクに移し2のオスとメスの2大人のゼブラフィッシュ（、3〜6ヶ月、健康的な実験室アブ・ストレイン）。バリアで男性と女性を分離します。翌朝、タンクの水をリフレッシュし、それらが交尾できるようにするために障壁を取り除きます。受精後1〜2時間、100ミリメートルペトリ皿に卵を収集します。 </li><li>初期の開発期間（4日間）の間に28.5°Cで40 mLの胚培地（EM）と100mmのプレートで約40胚を保ち、一日二回EMをリフレッシュ。 1 Lタンク（300 mLのシステム水40幼虫）に幼虫を移し、5 DPF後生ワムシ（全電源、一日二回）で供給。10 DPFの後にライブブラインシュリンプの代わりに、ワムシの食事を養います。 21 DPF 14に再循環水システムの中に魚を転送します。 </li><li> 17 DPF又は25 DPFまで再循環水システムにおける幼虫ゼブラフィッシュを上げます。明/暗サイクルを保証するシステムの水の14/10時間およびpH値は約7.2です。 17 DPF（5.5 6.8ミリメートル）の体長を測定し、25 DPF（8〜11 mm）の幼虫22。 </li></ol></li><li>解剖2％寒天プレートを準備します。 <ol><li> 200mLの滅菌水に4gの寒天を加えます。それが透明になるまでマイクロ波オーブン中で混合物を加熱します。 </li><li>次いで、直径60mmのペトリ皿（プレートのおよそ1/3量）にそれを注ぎ、約15分間、寒天溶液を冷却します。 4°Cで固化寒天プレートを保管してください。 </li></ol></li></ol> 2.プロトコール1：幼虫DPF 17及び25の生殖組織を分析<ol><li> 17 DPFの幼虫を麻酔するためにタンクに砕いた氷を追加します。転送30 mLの冷リンゲル液でペトリ皿冷却100mMの麻酔幼虫。完全に幼虫を麻酔するために、少なくとも15分間冷却リンゲル液中で培養した魚を保管してください。 </li><li>小さなプラスチックのスプーンで予冷した寒天プレートに幼虫を転送します。 10 mLの全体の魚の体を水没リンゲル液を冷却し、そっとその側にそれを置きます。 </li><li> 25X実体顕微鏡の視野の下で以下の操作を行います。安定化のためのピンセットで魚のトランクをクランプします。ピンセットの別のペアと心に肛門から縦方向に腹部をリッピング。静かに内臓を露出するために、本体の片側に皮膚や筋肉を取り除きます。 </li><li>慎重に浮き袋に腹器官の質量を削除します。浮き袋に添付生殖腺にダメージを与えることは避けてください。 </li><li>浮き袋と前部本体との間の接続を遮断します。優しく全体の浮き袋と生殖腺組織を引き出します。 注：ほとんどの場合S、17 DPFの性腺組織は、上皮組織とprotonephridiumを取り巻く含まれています。それらは容易に分離されていないが、25℃で容易に分離することができるDPF。生殖腺は、肛門に接続されています。肛門で相互に接続され、左右の生殖腺との接合は、はっきりと見えるであろう。 </li><li>慎重に浮き袋から生殖腺組織を分離し、周囲の脂肪組織をクリーンアップするためにピンセットを使用してください。 </li><li>直ちに200μLのリンゲル液を含有する予冷1.5mLの遠心管に、単離された生殖腺組織を移します。すべての生殖腺組織が幼虫から分離されるまで、チューブを氷上に保管してください。 </li><li> 2.7 - 手順2.1に記載したように25 DPFの幼虫の生殖腺を解剖。 </li></ol> 3.プロトコル2：孤立生殖腺組織の遺伝子発現を分析<ol><li>幼虫性腺組織からトータルRNAを抽出します。 <ol><li>新しいRNaseフリー1に分離された生殖腺組織を転送します。5mLのチューブ及びリンゲル液を除去します。 100μLの溶解液を追加します。組織が完全に溶解するまでボルテックス。 </li><li>製造業者の説明書に従って全RNA抽出手順を実行します。 </li><li>私はバッファ10倍DNアーゼの1/10量とRNA溶液にDNアーゼIの1μLを加え、穏やかに混合。 37°Cで20分間インキュベートします。 </li><li> RNA溶液に1/10量のDNase不活性化試薬を追加します。 70°Cで10分間インキュベートします。分光光度計を用いて全RNAの濃度を測定します。 -20℃で保管してください。 </li></ol></li><li>第一鎖cDNA合成を行います<ol><li>製造業者のプロトコルに従って第一鎖cDNA合成を実行するためにオリゴ（dT） - リンカープライマーを使用します。 20μLの総反応容量にRNAの1〜2μgのを追加。 </li><li> 45℃で90分間反応溶液をインキュベートします。 5分間70℃で加熱することによって反応を停止。 </li><li> cDNAに1μLのRNase Hを追加しますので、残留RNAを除去するためにlution。 〜13,000×gで10秒間静かに遠心混ぜます。 37°Cで20分間インキュベートします。 20μlのヌクレアーゼを含まない水を追加します。 -70℃で保管してください。 </li></ol></li><li>蛍光定量的PCRを行います<ol><li>蛍光色素でサイクラーシステム上で定量PCRを行います。 30秒、15秒、15秒、95℃で35サイクル、TM-5°C、68°C以下のPCRサイクル条件を使用します。次のようにプライマー配列を使用する：AMH（FWD：GTGGATGGCAGCAGTACGAC; REV：GCGGAGAGGTGGAAGAGAGAATG）、cyp19a1a（FWD：GTCCTGTTGTCTCCTACTGTCG; REV：CATTTGAGTTGAATATGATGCCCTG）、nanos3（FWD：GCTCGGTGTACGCCAAATCAACAT; REV：CCAAGTGAAAACACAACACCAGTGC）、 ヴァーサ （FWD：ATCGCATAGGAAGAACTGGACGCT; REV：CCAAGTGAAAACACAACACCAGTGC）とβアクチン （FWD：AGTGCGACGTGGACATCCGTA; REV：GCACTTCCTGTGGACGATGGA）19。 </li></ol></li></ol>

<ol>
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著者は、彼らが競合する金融利害関係を持たないことを宣言します。

ゼブラフィッシュは、強力なモデルとなっており、広範囲の開発と疾患関連研究に使用されています。例えば、脳、心臓、生殖腺、腎臓などのアダルトゼブラフィッシュにおける臓器の単離のための方法は、ウェル23、24、25を文書化されています。小型及び幼虫のゼブラフィッシュにおける生殖組織の動的再造形に、生殖...

野生のゼブラフィッシュ4染色体の主要な女性セックス行列は、1（ すなわち 、共通の実験室株）を紛失したり、飼いならされたゼブラフィッシュに変更されます。代わりに、彼らは、温度、低酸素症、食品の可用性と人口密度などの環境要因を伴う多遺伝子のセックス決意システムを持っています。ゼブラフィッシュのセックス開発の詳細なメカニズムは完全には理解されていません。プライマリ性別決意信号（単数または複数）/何であるか、そのようなゼブラフィッシュ性別決意が発生した場合のように基本的な質問は、であり、遺伝子は、生殖腺の変換の最初のステップは、3 2 に答えされずに残って調節います。 ゼブラフィッシュのセックス、開発の過程では、いくつかの重要な段階では認識されています。開発の初期段階では、4 HPF（時間後に受精）始原生殖細胞（始原生殖細胞）仕様を受け、から出発して生殖隆起に移動および増殖。 PGC番号と生殖細胞と体細胞間の相互の相互作用は、生殖腺の分化4のために重要です。 13 DPF（日受精後）では、生殖腺は、未分化段階にあります。 17のDPFすることにより、生殖腺は、将来、女性と男性の両方における双方向性卵巣へと発展します。卵巣からの精巣にアポトーシス依存遷移は21〜25 DPFから始まり、数週間継続することができます。 35のDPFにより、生殖腺の性別が決定されており、性別特異的配偶子生産は両方の卵巣に進行中であると5精巣、6、7。 現在まで、多様な候補遺伝子とセックス決意のメカニズムが提案されています。プロテオミクスとトランスクリプトーム解析は、性的二型表現で多くの遺伝子を単離したこれらの遺伝子は、ゼブラフィッシュ8に性分化を研究するために利用されてきました</sup&gt; 9、10。例えば、幼生ゼブラフィッシュでは、cyp19a1a遺伝子は特になく、精巣11,12における卵巣において発現されます。また、AMH遺伝子が弱く卵胞顆粒膜細胞で発現するが、強く精巣のセルトリ細胞13れます。対照的に、Vasa遺伝子は、連続的に適切な性腺マーカー14、15作り、女性と男性の両方ゼブラフィッシュの生殖細胞において発現されます。 性腺遺伝子発現レベルを調べることは、特に双電位卵巣ステージ3,9に性別決意と分化の分子機構を理解することが重要です。しかし、幼虫のゼブラフィッシュおよび対応小さな生殖腺のサイズが小さいgonadaの単離を複雑さらなる分子分析のためにL組織。以前の研究では、operculaと肛門の気孔16との間の解剖全体のトランク域を使用しました。この準備は、生殖腺を含むが、複数の組織や器官から構成されています。あるいは、脈などの生殖腺特異的GFP発現を有するトランスジェニック動物：EGFPの蛍光活性化細胞選別（FACS）およびレーザー捕捉マイクロ切開17、18 を介して、性腺組織単離のために使用しました。しかし、彼らの広範なアプリケーションが限られています。ここでは、17 DPFと25 DPFの幼虫のゼブラフィッシュからの性腺組織を分離するための簡単な手順について説明します。我々は、他の器官に対する生殖腺の位置を示し、周囲の組織から形態学的に無傷の生殖腺を分離します。我々はさらに、（例えばヴァーサとcyp19a1aなどの生殖腺特異的遺伝子は高度に定量的PCRを介してトランク組織と比較して、単離された生殖腺で発現される表示します定量PCR）分析。本プロトコルは、それによって性腺組織19のその後の分子分析を可能にする、幼生ゼブラフィッシュから生殖腺特異的遺伝子の同定、単離、RNAの精製及び増幅を可能にします。

<table><tbody><tr><td>Cell culture dish 100 mm</td><td>Corning</td><td>430167</td><td>For embryo incubation</td></tr><tr><td>20x EM</td><td></td><td></td><td>For a 1 L needed: add 17.5 g NaCl, 0.75 g KCl and 2.9 g CaCl&amp;#183;2H&lt;sub&gt;2&lt;/sub&gt;O; then add 0.41 g KH&lt;sub&gt;2&lt;/sub&gt;PO4, 0.412 g Na&lt;sub&gt;2&lt;/sub&gt;HPO4 anhydrous and 4.9 g MgSO&lt;sub&gt;4&lt;/sub&gt;&amp;#183;7H&lt;sub&gt;2&lt;/sub&gt;O.</td></tr><tr><td>1x EM</td><td></td><td></td><td>Dilute 20x EM in distilled water</td></tr><tr><td>AGAROSE G-10</td><td>Gene</td><td>121985</td><td>For preparing the 2% agar plates</td></tr><tr><td>Trizol Reagent</td><td>Invitrogen</td><td>15596-026</td><td>For RNA isolation</td></tr><tr><td>Meter glass</td><td>Shen Bo</td><td>250 ml</td><td>For preparing the 2% agar plates</td></tr><tr><td>Microwave Oven</td><td>Midea</td><td>M1-211A</td><td>For heating the AGAR</td></tr><tr><td>Tweezer DUMONT#5INOX</td><td>World Precision Instrument</td><td>500341</td><td>For dissection</td></tr><tr><td>Stereomicroscope</td><td>Motic</td><td>SMZ168</td><td>For dissection</td></tr><tr><td>Pure water equipment</td><td>Millipore</td><td></td><td></td></tr><tr><td>Ringer&amp;rsquo;s solution</td><td></td><td></td><td>For a 1 L needed: Add 6.78 g NaCl, 0.22 g KCl, 0.26 g CaCl&lt;sub&gt;2&lt;/sub&gt; and 1.19 g Hepes; then fill to 1 L; Adjust pH to 7.2. Sterilize by filtration and keep in an autoclaved clear polycarbonate container.</td></tr><tr><td>Transfer pipette</td><td>Samco</td><td>202, 204</td><td></td></tr><tr><td>Metal bath</td><td>QiLinbeier</td><td></td><td>Model GL-150</td></tr><tr><td>Microscope</td><td>Leica&amp;nbsp;</td><td>M205 FA</td><td>For photomicrograph</td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5417R</td><td></td></tr><tr><td>Micro Scale RNA Isolation Kit</td><td>Ambion</td><td>AM1931</td><td>For RNA isolation from gonad tissues</td></tr><tr><td>Dnase I</td><td>Sigma</td><td>AMPD1-1KT</td><td>For DNA digestion in the RNA solution</td></tr><tr><td>RevertAid First Strand cDNA Synthesis Kit</td><td>Thermo Scientific</td><td>#K1631</td><td>For first-strand cDNA synthesis</td></tr><tr><td>Rnase H</td><td>Thermo Scientific</td><td>#EN0202</td><td>For digesting the residual RNA in the cDNA solution.</td></tr><tr><td>SYBR Green Realtime PCR Master Mix</td><td>TOYOBO</td><td>QPK-201</td><td>This product is a Taq DNA polymerase-based 2x master mix for real-time PCR and&amp;nbsp; applicable for intercalation assay with SYBR Green I.</td></tr><tr><td>Spectrophotometer</td><td>Ne Drop</td><td>OD-2000+</td><td>Measuring the concentration of the total RNA</td></tr><tr><td>Mastercycler</td><td>Eppendorf</td><td>AG 22331 Hamburg</td><td>gene expression profiling</td></tr></tbody></table>

dissection of larval zebrafish gonadal tissue

野生のゼブラフィッシュは、ZZ / ZWセックス決意システムを有するが、飼いならされたゼブラフィッシュは、性染色体を失っています。彼らは、ゲノム全体に分布するいくつかの遺伝子は、まとめて、個々の魚の性同一性を決定多遺伝子性別決意システムを利用します。現在、生殖腺の発達の調節に関与する遺伝子とその仕組みは、とらえどころのないまま。通常、生殖腺組織を分離することは、セックスの発生過程を調べるための最初のステップです。ここでは、ゼブラフィッシュの幼虫DPF 17 DPF（日受精後）と25からの性腺組織を分離するための手順を提示します。単離された生殖腺組織は、その後、形態学および遺伝子発現プロファイリングによって調べることができます。

生殖腺の解剖は、アブ・ストレイン幼虫のゼブラフィッシュで行われました。 図1は、17 DPF、25 DPFにおける幼虫のゼブラフィッシュの典型的な性腺組織を示しています。まず、皮膚や腹部の片側の筋肉は内臓を露出するように切断されます。臓器の質量を除去した後、一緒に生殖腺を有する水泳膀胱トランクに残ります。生殖腺は、水泳膀胱（ <...

Watch this Scientific Journal Video about Dissection of Larval Zebrafish Gonadal Tissue at JoVE.com

Dissection of Larval Zebrafish Gonadal Tissue

ここでは、ゼブラフィッシュの性分化とメンテナンスの調査を容易にするであろう幼虫のゼブラフィッシュの生殖腺組織を単離するためのプロトコルを提示します。

幼生ゼブラフィッシュ性腺組織の解剖

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

dissection-of-larval-zebrafish-gonadal-tissue

Research

JoVE Journal

Developmental Biology

10.2K ビュー.  Fudan University. ここでは、ゼブラフィッシュの性分化とメンテナンスの調査を容易にするであろう幼虫のゼブラフィッシュの生殖腺組織を単離するためのプロトコルを提示します。 

ビデオ: Dissection of Larval Zebrafish Gonadal Tissue

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

解剖やゼブラフィッシュ胚の横方向の取付：脊髄発達の解析

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

神経科学

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

アダルトゼブラフィッシュからの臓器の解剖

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

生物学

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

幼虫のゼブラフィッシュにおける脊髄切断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, germ cell development, piRNA biology, and niche formation. Here, we present a dissection technique to live-image the gonad ex vivo during a period when in vivo live-imaging is highly ineffective. This protocol outlines how to transfer embryos to an imaging dish, choose appropriately-staged male embryos, and dissect the gonad from its surrounding tissue while still maintaining its structural integrity. Following dissection, gonads can be imaged using a confocal microscope to visualize dynamic cellular processes. The dissection procedure requires precise timing and dexterity, but we provide insight on how to prevent common mistakes and how to overcome these challenges. To our knowledge this is the first dissection protocol for the Drosophila embryonic gonad, and will permit live-imaging during an otherwise inaccessible window of time. This technique can be combined with pharmacological or cell-type specific transgenic manipulations to study any dynamic processes occurring within or between the&#160;cells in their natural gonadal environment.

Dissection and Live-Imaging of the Late Embryonic Drosophila Gonad

Here, we provide a dissection protocol required to live-image the late embryonic Drosophila male gonad. This protocol will permit observation of dynamic cellular processes under normal conditions or after transgenic or pharmacological manipulation.

後期胚 性ショウジョウバエ生殖腺 の解剖とライブイメージング

The Drosophila melanogaster male embryonic gonad is an advantageous model to study various aspects of developmental biology including, but not limited to, ...

発生生物学

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

ゼブラフィッシュ胚眼の組織の顕微解剖

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

生きているゼブラフィッシュ幼虫の眼球除去による神経支配依存性の成長と視覚系の発達を調べる

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...

ゼブラフィッシュ幼生の腸細胞をプロセッシングし、シングルセル RNA シーケンシングを行う(英語)

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live,&#160;viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

著者スポットライト:ゼブラフィッシュ幼生の腸内細胞の単離と分析による胃腸発生のトランスクリプトーム的側面の調査 

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

シングルセルRNAシーケンシングのためのゼブラフィッシュ幼生からの腸管分離

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

幼生ゼブラフィッシュ性腺組織の解剖

要約

さらに動画を探す

この動画の章

解剖やゼブラフィッシュ胚の横方向の取付：脊髄発達の解析

アダルトゼブラフィッシュからの臓器の解剖

幼虫のゼブラフィッシュにおける脊髄切断

後期胚性ショウジョウバエ生殖腺の解剖とライブイメージング

ゼブラフィッシュ胚眼の組織の顕微解剖

生きているゼブラフィッシュ幼虫の眼球除去による神経支配依存性の成長と視覚系の発達を調べる

シングルセルRNAシーケンシングのためのゼブラフィッシュ幼生からの腸管分離

シングルセルRNAシーケンシングのためのゼブラフィッシュ幼生からの腸管分離

シングルセルRNAシーケンシングのためのゼブラフィッシュ幼生からの腸管分離

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ

幼生ゼブラフィッシュ性腺組織の解剖

要約

さらに動画を探す

この動画の章

解剖やゼブラフィッシュ胚の横方向の取付：脊髄発達の解析

アダルトゼブラフィッシュからの臓器の解剖

幼虫のゼブラフィッシュにおける脊髄切断

後期胚 性ショウジョウバエ生殖腺 の解剖とライブイメージング

ゼブラフィッシュ胚眼の組織の顕微解剖

生きているゼブラフィッシュ幼虫の眼球除去による神経支配依存性の成長と視覚系の発達を調べる

シングルセルRNAシーケンシングのためのゼブラフィッシュ幼生からの腸管分離

シングルセルRNAシーケンシングのためのゼブラフィッシュ幼生からの腸管分離

シングルセルRNAシーケンシングのためのゼブラフィッシュ幼生からの腸管分離

幼虫の心臓の解剖、少年と大人ゼブラフィッシュ、ゼブラフィッシュ

後期胚性ショウジョウバエ生殖腺の解剖とライブイメージング