この作品は、MCMとTGの米国癌学会特別研究員＃PF - 05 - 041 - 01 - DDCにNIHの助成R01 HD050901によってサポートされていました。

<ol><li>男性のゼブラフィッシュは、メスの魚の順で解剖されます。解剖を始める前に、0.2％Tricaineで魚を麻酔し、15分間氷水中でインキュベーションすることで安楽死させる。 </li><li>軽くペーパータオル上で乾燥した魚を叩くと、解剖用マットの上に置くことによって始めます。外部から、ゼブラフィッシュは、単一の背、尾と鰭と対になった胸と骨盤のフィン（図1）を持ちます。 <img src="/files/ftp_upload/1717/1717fig1.jpg" alt="図1" /> 図1ラベル付きのフィンを持つ成人男性の魚。 </li><li>尾の肉厚部を介して解剖用マットに魚と眼窩の腹側部分を固定。 </li><li>しりびれのちょうど前の魚の腹の皮膚をスニップ。しりびれから蓋に腹（ギル以上ハードカバー）（図2、ステップ1）に沿って皮膚と基礎となる筋肉をカット。 <img src="/files/ftp_upload/1717/1717fig2.jpg" alt="図2" /> 図2皮膚や内臓を公開するために体壁筋を取り除くのステップ。 </li><li>次に、胸帯（フィンの基部の厚さ、骨の多い地域）（図2、ステップ2）を含めて、蓋と胸びれを取り外します。ダウン尻鰭（図2、ステップ3）にして魚の側面に沿って、現在露出鰓後方の上から始まり、皮膚の基礎となる筋肉をカット。 </li><li>慎重に魚の側面から皮膚や基礎となる筋肉を取り除く。内臓の多くは、現在（図3）表示されます。 <img src="/files/ftp_upload/1717/1717fig3.jpg" alt="図3" /> 図3。体壁と筋肉を持つ成人男性の魚は内臓の可視化を可能に削除。 </li><li>精巣は、背側体壁に接続されている長い、白、対になった器官である。精巣を削除し、PBSの皿に入れてください。 spermatagoniaから精子への発展途上の生殖細胞のさまざまな段階（レアルら、2009）で嚢胞を含む精細管（図4）、可視化するために反射光で精巣を調べます。 <img src="/files/ftp_upload/1717/1717fig4.jpg" alt="図4" /> 図4精巣の解剖。白い丸みを帯びた構造は精細管です。 </li><li>次に、魚の体腔から胃腸系を削除します。肝臓はその大きさ、ローブ形態、黄褐色がかった色、そして豊富な血管新生によって識別することができます。明るい赤を表示される胆嚢、緑色の半透明の液体で満たされた嚢、および脾臓は、内臓の中にあります。 </li><li>臓器の残りの部分から腸を分離し、それを差し伸べる。腸の前方、中間、および後部領域は上皮ひだの高さ（Wallaceら、2005）によって定義されています。 </li><li> PBSで腸の一部を配置し、透過光を用いて上皮ひだを観察。 </li><li>次に、浮き袋を調べます。浮き袋は、ウェーバーの装置（フィニーら、2006）を介して内耳に接続されている空気圧ダクト、および前房、を経由して食道に接続されている後房、から構成されています。 </li><li>浮き袋を取り外して廃棄。魚の固定を解除し、再度ピンに腹側を上に背側体壁に沿って配置されている腎臓を、分析する。腎臓は背側大動脈と色素細胞に関連付けられている半透明のピンクの構造体です。腎臓は、頭部、ボディ、および尾部の領域（図5）に分かれています。腎臓の一部を摘出し、PBSに入れてください。腎尿細管を明らかにするために腎臓の組織を離れていじめる。 <img src="/files/ftp_upload/1717/1717fig5.jpg" alt="図5" /> 図5。頭部、ボディ、および背側体壁に沿って尾の腎臓の位置。 </li><li>メスの魚を解剖。前述のように、氷水で魚を安楽死させると解剖マットにそれを固定する前に乾燥したそれをなでる。以前に示したように魚の側面から皮膚を取り除きます。卵巣は、血管形成の卵巣間膜によって体腔に一時停止されているbilobed構造です。 </li><li>卵巣の一葉を除去、PBSに入れ、透過光でそれを調べる。卵母細胞は微細な針を使用して離れてからかったし、（図6、セルマンら、1993）ステージングできます。 I期の卵母細胞は約10です - 大きさと半透明の150ミクロン。大きさは350ミクロンと皮質顆粒の出現によって定義 - II期の卵母細胞は、約150です。 III期の卵母細胞は350です - 大きさで750ミクロンと卵黄の蓄積による不透明です。成熟期IVおよびVの卵母細胞は半透明であり、一般に最近交配した雌の​​卵巣には見られない。 <img src="/files/ftp_upload/1717/1717fig6.jpg" alt="図6" /> 図6。ライブステージI、II、およびIIIの卵母細胞は下に観察さ透過光で顕微鏡を解剖。 </li><li>次に、魚から心臓を解剖。心臓は後部と鰓に腹側に位置しています。心と周囲の組織のすべてを切断し、PBSに置くことで始める。慎重に繊細なアトリウムを傷つけないように注意しながら、心臓周辺の組織を離れて分析する。 </li><li>ハートビートを監視するためにリンゲル液中に切除した心臓を置きます。 心房、心室、および動脈球（図7、Huら、2001）を特定します。 <img src="/files/ftp_upload/1717/1717fig7.jpg" alt="図7" /> 図7。成人心臓を解剖。 </li><li>魚から脳を削除して解剖を完了します。魚を固定解除し、かみそりの刃で頭を最初に取り外します。鉗子で頭蓋骨の腹側から可能な限り多くの軟組織のように取り外します。小さな春のはさみを使って目を削除します。頭蓋骨を開き、脳の腹側から骨を除去破る。今PBSの皿に頭を置いて、脳の背側から皮膚と頭蓋骨の骨を取り除く。 </li><li>嗅球、終脳、手綱、視蓋、小脳および延髄（。。シリング、2002図8、Wullimannら、1996）を特定します。 <img src="/files/ftp_upload/1717/1717fig8.jpg" alt="図8" /> 図8。ディテールに秘められた大人のゼブラフィッシュの脳。トップへ前背ビュー、。 </li></ol>

<ol>
	<li>Finney, J. L., Robertson, G. N., McGee, C. A., Smith, F. M., Croll, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Autonomic+Innervation+of+the+Swim+Bladder+in+the+Zebrafish+(Danio+rerio).">Structure and Autonomic Innervation of the Swim Bladder in the Zebrafish (Danio rerio).</a> J Comp Neurol. 495, 587-606 (2006).</li><li>Hu, N., Yost, H. J., Clark, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+morphology+and+blood+pressure+in+the+adult+zebrafish.">Cardiac morphology and blood pressure in the adult zebrafish.</a> Anat Rec. 264, 1-12 (2001).</li><li>Leal, M. C., Cardoso, E. R., N&#243;brega, R. H., Batlouni, S. R., Bogerd, J., Fran&#231;a, L. R., Schulz, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+stereological+evaluation+of+zebrafish+(Danio+rerio)+spermatogenesis+with+an+emphasis+on+spermatogonial+generations.">Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations.</a> Biol Reprod. 81, 177-187 (2009).</li><li>Schilling, T. F., Nusslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+morphology+of+larval+and+adult+zebrafish.">The morphology of larval and adult zebrafish.</a> Zebrafish: A Practical Approach (The Practical Approach Series). , (2002).</li><li>Selman, K., Wallace, R., Sarka, A., Qi, X. <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+oocyte+development+in+the+Zebrafish,+Brachydanio+rerio.">Stages of oocyte development in the Zebrafish, Brachydanio rerio.</a> J. Morphol. 218, 203-224 (1993).</li><li>Wallace, K. N., Akhter, S., Smith, E. M., Lorent, K., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+growth+and+differentiation+in+zebrafish.">Intestinal growth and differentiation in zebrafish.</a> Mech Dev. 122, 157-173 (2005).</li><li>Wullimann, M. F., Rupp, B., Reichert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroanatomy of the zebrafish brain : a topological atlas. , (1996).</li></ol>

動物実験は、ペンシルベニア州動物実験の大学および使用委員会によって定められたガイドラインおよび規制に準拠して行った。

試薬： 0.2パーセントTricaine（3 -アミノ安息香酸エチル） 200mgのtricaine粉 97.9ミリリットルDDの水 〜1mlのMトリス（pH 9） pHを7.0に調整する リン酸緩衝食塩水（PBS） 4.0グラムのNaCl 0.1グラムのKCl 100ミリリットルの0.1 M PO 4緩衝液、pH7.3に 150ミリリットルのdH 2 O リンガーソリューション 6.7グラムのNaCl 0.2グラムのKCl 0.2グラムのCaCl 2 1.2グラムのHepes 1 LH 2 O 7.2にpHを調整する設備： マットと解剖皿 電子顕微鏡学 カタログ＃70540 Vannas春はさみ ファイン科学ツール カタログ＃91500〜09

dissection of organs from the adult zebrafish

過去20年間で、ゼブラフィッシュは、脊椎動物の発生や疾患を理解するための強力なモデル生物となっています。胚および幼虫の実験的分析は広範であり、形態が十分に文書化されているが、一緒に大人と一緒に作業するためのテクニックを持つ成人のゼブラフィッシュの解剖学と成人の構造や臓器の開発の研究の説明は、不足している。幼虫の臓器は、その全体的な構造、形態、及び成人への移行幼虫の時の解剖学的位置に大きな変化を受ける。内部的に、臓器が巨大な成長とリモデリングを受ける間、外部から、透明な幼虫は、その特徴的な大人のストライプpigmentパターンと対になった骨盤のフィンを開発しています。さらに、両性能の生殖腺の原基が精巣か卵巣のどちらかに開発しています。このプロトコルは、成体の器官の多くを識別し、脳、生殖腺、消化器系、心臓、および成人のゼブラフィッシュの腎臓の解剖のための方法を示しています。解剖の臓器は、in situハイブリダイゼーション、免疫組織化学、組織学、RNAの抽出、タンパク質解析、および他の分子技術のために使用することができます。このプロトコルは、幼虫の臓器のリモデリング、成体の器官系の成人およびその他の調査への特定の器官の形態形成を含むようにゼブラフィッシュでの研究の広がりを支援します。

Watch this Scientific Journal Video about Dissection of Organs from the Adult Zebrafish at JoVE.com

Dissection of Organs from the Adult Zebrafish

このプロトコルは、成人のゼブラフィッシュから臓器を特定し、解剖するための手順を説明します。

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 ...

dissection-of-organs-from-the-adult-zebrafish

Research

JoVE Journal

Biology

103.9K ビュー.  University of Pennsylvania-School of Medicine. このプロトコルは、成人のゼブラフィッシュから臓器を特定し、解剖するための手順を説明します。

ビデオ: Dissection of Organs from the Adult Zebrafish

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

アダルトゼブラフィッシュの内耳感覚上皮のグロスとファイン解剖（ゼブラフィッシュ）

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

生物学

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

成体マウスから海馬歯状回の解剖

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

アダルトゼブラフィッシュへの腹腔内注射

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

大人からOenocytesの解剖キイロショウジョウバエ</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

直接アダルトゼブラフィッシュの腎臓への細胞の移植

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

アダルトゼブラフィッシュ腎臓の解剖

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

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 ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

アダルトゼブラフィッシュ網膜にモルフォの in vivoエレクトロポレーションに

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

リジェネアダルトゼブラフィッシュテールフィンにMorpholinos の in vivoエレクトロポレーションで

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavagingアダルトゼブラフィッシュ

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...