この手順では、ビルジェフリーの研究室で洞窟の魚のために開発された大部分は移植技術に従います。

パート1：胚を準備このプロトコルでは架空ゼブラフィッシュレンズ変異を示すためJJ なxyシンボルを使用します。 <ol><li>ゼブラフィッシュ株は14H light/10h暗サイクル28.5 Cで標準魚ファシリティ条件で維持さ。 </li><li>夕方、場所雄とゼブラフィッシュひずみJJ xyの雌における：AB / TU;互いにからそれらを分離するデバイダ持つタンクにおけるTG（MGFP）。この十字架の子孫はすべて組織におけるGFP導入遺伝子を発現するであろう、およびドナーとして使われます。パラレルで、非トランスジェニック野生型動物間交雑をセットアップする同じ手順を使用。この実験の目的に応じ、一つドナーまたはホストとしてJJ なxy座で野生型動物を交差できます。 </li><li>光が朝でターンオン後1時間以内、メイトへ魚を許可する仕切りを取り外します。 </li><li> 15-30分間が100 × 15ミリメートルシャーレで胚を収集後、それらを掃除、と培地（卵水を）変更。 </li><li>希望時間まで28.5 Cインキュベーターで胚キープ。 </li><li>するとき移植用準備、鋭い鉗子の二対で手動絨毛膜を取り外し、および30分間カルシウム-フリーZFRで0.2％EDTAで胚をインキュベート。 </li></ol>パート2：解剖針の調製<ol><li> ;とドナーレンズを移動とホストに挿入する使わ鈍針、レンズを取り巻く組織をカット絨毛膜を削除、およびアガロースからリリース胚に使われる尖った一つ：移植手順は針の二種類を必要。ここではこれら針を作る方法を紹介します。 </li><li>最初、パスツールピペットの先端はダイヤモンドナイフを使っ断たれるなりません。その後その長の約半分がそれ内側なるようパスツールピペットチップにキャピラリーガラスを挿入。これは容易次ステップでガラスにそれを溶融によってタングステンワイヤを動けなくする行います。 </li><li>その後キャピラリーの開口端に細いタングステン線を挿入。ブンゼンバーナーとともにワイヤーにわたってガラスをメルト。手で針の他端をねじれながら鉗子を使用、金属線とともに着実ガラス終わりをホールド。軟化したガラスは場所にを把持金属周りスパイラルすべき。 </li><li>もし鈍先端針を作っている場合、このは手順の終わりです。シャープ針を作成する、非常細かい先端が作成れるよう金属をオフ燃焼、1から1.5分間ブンゼンバーナーにわたってタングステンワイヤーをホールド。 microscropy.Itによって先端の品質が移植前直ちに数秒間火炎における両方針の滅菌に良いアイデアですチェック。 </li></ol>パート3：試薬の調製<ol><li>カルシウム-フリーゼブラフィッシュリンゲル（ZFR）ソリューションズ（116 mMのNaCl、2.9 mMののKCl、5mMのHEPES、pH7.2を） </li><li>カルシウム-フリーゼブラリンゲルソリューション（液pH7.2）における0.2％EDTA </li><li>カルシウム-フリーZFRにおける1.2％アガロース（低融点） </li><li>カルシウム-フリーZFRの0.2％アガロース（低融点） </li><li>リットルあたり胚培地は（pH7.0の、含ま：10ミリリットルハンクスソリューション＃1、1ミリリットルハンクスソリューション＃2、10ミリリットルハンクスソリューション＃4、10ミリリットルハンクスソリューション＃5、0.35 gの重炭酸ナトリウム、2M塩酸の300 UL、ペニシリン-ストレプトマイシン500,000をゼブラフィッシュブック （オレゴンプレス大学、2000）におけるとしてU）。 </li><li>タングステンワイヤー、金メッキ、0.1ミリメートル直径、アルファAesar </li><li>毛細管、1.0ミリメートル直径、世界プレシジョンインスツルメンツ</li></ol>パート4：移植手続き<ol><li>プラスチック遠心管に、水浴40 Cに予熱カルシウム-フリーZFRで1.2％低融点アガロースを準備。 </li><li>希望ステージ（例えば30 HPF、）で、ゆっくり遠心管にそれらを追放、およびゆっくり遠心管にそれらを追放と約5秒間インキュベート、パスツールピペットで胚を占有。ドナーとホストは別途インキュベートれるべき。 </li><li>ホストとドナーが別個保つ、ピペットとともに、遠沈管から​​1.2％アガロースで胚を占有と100ミリメートルポリスチレンシャーレ内二つの平行行でそれらを手配。ゆっくりと注意深くそれらをペッティングによって、最も胚はアガロースにおける彼ら両側にうそなります。あなたはアガロースが固化前眼が上向きれるよう鈍針を使っこの位置にうそしないもの向きを変更する必要が。 </li><li>固化に1.2％アガロースを待ちます、および、0.2％低融点アガロース溶液でそれをオーバーレイ。 </li><li>シャープ針を使用、目の周りいかなるアガロースを取り外し、および非常注意深く小さなストロークを使っホストとドナー胚からレンズを切り取る。ようそれを引き裂くはなくレンズするちょうど十分近いカットする必ず確認、ならずのでホスト眼の残りからすぎる組織を削除しない。 </li><li>かつてフリー、レンズは培地でフロートします。鈍針を使用、注意深くドナーをプッシュレンズは、単にホストのレンズが正常になる場所の位置の上にして、鈍針で目の中に、押し下げてください。ホストのレンズは破棄されることがあります。 </li><li> 30〜60分間、1.2％アガロースでドナーとホスト胚のままにして、鋭い針を用いてアガロースからそれらを解放し、胚の培地で24ウェルプレートにそれらを転送する。それぞれの胚には、別々の井戸を占有する必要があります。正しくので、ホストがどのドナーに対応するかを明確であることを井戸にラベルを付けることを確認してください。 </li><li> 28.5℃でインキュベートするドナー由来のレンズは、GFP蛍光に基づいて、同じ動物の逃れた側のホストのレンズと区別することができる。セクションはまた、レンズのサイズを測定するために、それが適切に区別するかどうかを判断するために行うことができます。 </li></ol><img src="/files/ftp_upload/1258/1258fig1.jpg" alt="図1" /> 図。レンズ移植のために使用されて削られた針の1低倍率画像。キャピラリーガラス（B）は、パスツールピペット（A）に挿入され、細いタングステンワイヤ（C）は、キャピラリーの開放端に挿入されます。ワイヤー上のガラスは、ブンゼンバーナーで柔らかくします。あなたの手でパスツールピペットのもう一方の端をねじりながら、鉗子を用いて、金属線で安定したガラスの端を保持する。軟化したガラスは、金属の周りスパイラル、所定の位置に把持すべき。 <img src="/files/ftp_upload/1258/fig2 1258.png" alt="" width="600" height="204" /> 図。レンズ移植のために使用される注射針の二種類の2のヒント。 <img src="/files/ftp_upload/1258/1258fig3.jpg" alt="図3" /> 図。 3胚は30hpfでレンズ移植を施行した。示すように、ドナー胚（A、B）、移植レンズ（C、D）、およびホスト胚（E、F）の制御側でのホストの目です。示されるように各眼は透過光およびUV照射の両方で表示されます。写真は48 HPFで撮影したもの。 Q01 [9]トランスジェニックラインは、この実験に用いた。

<ol>
	<li>Thut, C. J., Rountree, R. B., Hwa, M., Kingsley, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+large-scale+in+situ+screen+provides+molecular+evidence+for+the+induction+of+eye+anterior+segment+structures+by+the+developing+lens.">A large-scale in situ screen provides molecular evidence for the induction of eye anterior segment structures by the developing lens.</a> Dev Biol. 231, 63-76 (2001).</li><li>Yamamoto, Y., Jeffery, W. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+role+for+the+lens+in+cave+fish+eye+degeneration.">Central role for the lens in cave fish eye degeneration.</a> Science. 289, 631-633 (2000).</li><li>Easter, S. S., Nicola, G. N. <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+vision+in+the+zebrafish(Danio+rerio).">The development of vision in the zebrafish(Danio rerio).</a> Dev Biol. 180, 646-663 (1996).</li><li>Schmitt, E., Dowling, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+eye+morphogenesis+in+the+zebrafish.+Brachydanio+rerio.">Early eye morphogenesis in the zebrafish. Brachydanio rerio.</a> J.comp. Neuro. 344 (4), 532-542 (1994).</li><li>Malicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Harnessing+the+power+of+forward+genetics--analysis+of+neuronal+diversity+and+patterning+in+the+zebrafish+retina.">Harnessing the power of forward genetics--analysis of neuronal diversity and patterning in the zebrafish retina.</a> Trends Neurosci. 23 (11), 531-541 (2000).</li><li>Malicki, J., Pujic, Z., Thisse, C., Thisse, B., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Forward+and+reverse+genetic+approaches+to+the+analysis+of+eye+development+in+zebrafish.">Forward and reverse genetic approaches to the analysis of eye development in zebrafish.</a> Vision Res. 42 (4), 527-533 (2002).</li><li>Avanesov, A., Malicki, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Approaches+to+study+neurogenesis+in+the+zebrafish+retina.">Approaches to study neurogenesis in the zebrafish retina.</a> Methods Cell Biol. 76, 333-384 (2004).</li><li>Vihtelic, T. S., Hyde, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+mutagenesis+yields+eye+morphological+mutants+with+retinal+and+lens+defects.">Zebrafish mutagenesis yields eye morphological mutants with retinal and lens defects.</a> Vision Res. 42, 535-540 (2002).</li><li>Godinho, L., Mumm, J. S., Williams, P. R., Schroeter, E. H., Koerber, A., Park, S. W., Leach, S. D., Wong, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+of+amacrine+cell+neurites+to+appropriate+synaptic+laminae+in+the+developing+zebrafish+retina.">Targeting of amacrine cell neurites to appropriate synaptic laminae in the developing zebrafish retina.</a> Development. Nov 132, 5069-5079 (2005).</li></ol>

いくつかのステップは、レンズの移植中に特別な注意が必要です。 <ol><li>針の先鋭化：これは、レンズの移植を実施する前にいくつかの針を準備すると良いでしょう、と針の先端はできるだけ薄くする必要があります。厚い針を癒すために目に障害を引き起こし、ためにより広い直径のより多くの組織を引き裂くだろう。 </li><li>胚の配置：彼らが両側に横たわっているように右移植前に?...

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lens transplantation in zebrafish and its application in the analysis of eye mutants

レンズは、眼杯[1,2]の開発において重要な役割を果たしている。モデル生物としてゼブラフィッシュを用いて、レンズの開発についての質問に対処することができます。ゼブラフィッシュは、小型サイズ、高い繁殖力、短いライフサイクル、およびケアの容易さなど、いくつかの有利な特性に起因する遺伝学的研究に有用です。レンズの開発は、ゼブラフィッシュで急速に発生します。 72 HPFにより、ゼブラフィッシュのレンズは、機能的に成熟している[3]。豊富な遺伝と分子資源は、ゼブラフィッシュの研究をサポートするために用意されています。さらに、他の脊椎動物のものとゼブラフィッシュアイの類似性は、人間の欠陥の優れた動物モデルとしての使用（4）〜（7）の基準を提供します。いくつかのゼブラフィッシュの突然変異体は、いくつかのケースでは、レンズティッシュ[8]の完全な変性につながる細胞死、の高いレベルを含む、レンズの異常を示す。 レンズの異常は、本質的な原因または周囲の組織と欠陥の相互作用によるものかどうか判断するために、野生型の目に変異体レンズの移植が行われている。ファイアーポリッシュ金属針を使用して、変異体または野生型レンズは、慎重にドナー動物から解剖し、ホストに転送されます。野生型と変異体の組織を区別するために、トランスジェニック系統をドナーとして使用されます。この行は、レンズを含めて、全ての組織において膜結合型GFPを発現する。この移植法は、ゼブラフィッシュレンズの変異体の研究に不可欠なツールです。

Watch this Scientific Journal Video about Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants at JoVE.com

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

レンズ開発は、他の組織との相互作用を伴います。いくつかのゼブラフィッシュの眼の変異体が異常に小さいレンズの大きさによって特徴付けられる。ここでは、この表現型は、レンズを囲む組織との本質的な原因や欠陥の相互作用によるものかどうかを判断するためにレンズの移植実験を示しています。

レンズゼブラフィッシュでの移植とアイ変異体の解析への応用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

lens-transplantation-zebrafish-its-application-analysis-eye

Research

JoVE Journal

Biology

11.0K ビュー.  The Second Teaching Hospital of Jilin University. レンズ開発は、他の組織との相互作用を伴います。いくつかのゼブラフィッシュの眼の変異体が異常に小さいレンズの大きさによって特徴付けられる。ここでは、この表現型は、レンズを囲む組織との本質的な原因や欠陥の相互作用によるものかどうかを判断するためにレンズの移植実験を示しています。

ビデオ: Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

アダルトゼブラフィッシュの全腎骨髄の移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

生物学

Preparation and Culture of Rat Lens Epithelial Explants for Studying Terminal Differentiation

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  Following division, these cells migrate posteriorly, where FGF diffusing from the retina induces them to differentiate into a posterior array of elongated lens fiber cells, which compose the bulk of the lens.  Differentiation of lens epithelial cells into lens fibers can be induced in vitro by culturing explants of the central region of the anterior epithelium in the presence of FGF-2.  Explants are prepared from lenses of neonatal rats by removing the lens from the eye and grasping the lens capsule on the posterior side with dissecting tweezers.  The posterior capsule is then gently torn open and pressed down into the plastic bottom of a tissue culture dish. The peripheral regions of the explant are removed with a scalpel and the central area is then cultured in the presence of 100ng/ml FGF-2 for as long as 2-3 weeks, depending on the parameters to be studied.  Since epithelial cells in cultured explants differentiate in approximate synchrony over a period of days to weeks, the time course of signaling and gene expression can be determined using molecular, biochemical, and pharmacological techniques.  Immunofluorescence microscopy is a powerful adjunct to these methods as it demonstrates the subcellular localization of proteins of interest and can reveal the physiological consequences of experimental manipulations of signaling pathways.

Explants of the central region of rat lens epithelia differentiate synchronously when cultured in the presence of FGF-2. Immunofluorescence microscopy of such cultures can provides novel information about gene expression and signaling events associated with terminal differentiation.

最終分化を研究するための準備とラットの水晶体上皮外植片の培養

The anterior surface of the ocular lens is covered by a monolayer of epithelial cells, which proliferate in an annular zone underlying the ciliary body.  ...

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti. 

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the inner ear (fig 1). Hair cells in the developing cochlea, which are the mechanosensory cells of the auditory system, are aligned in one row of inner hair cells and three (in the base and mid-turns) to four (in the apical turn) rows of outer hair cells that span the length of the organ of Corti. Hair cells transduce sound-induced mechanical vibrations of the basilar membrane into neural impulses that the brain can interpret. Most cases of sensorineural hearing loss are caused by death or dysfunction of cochlear hair cells.
An increasingly essential tool in auditory research is the isolation and in vitro culture of the organ explant 1,2,9. Once isolated, the explants may be utilized in several ways to provide information regarding normative, anomalous, or therapeutic physiology. Gene expression, stereocilia motility, cell and molecular biology, as well as biological approaches for hair cell regeneration are examples of experimental applications of organ of Corti explants.
This protocol describes a method for the isolation and culture of the organ of Corti from neonatal mice. The accompanying video includes stepwise directions for the isolation of the temporal bone from mouse pups, and subsequent isolation of the cochlea, spiral ligament, and organ of Corti. Once isolated, the sensory epithelium can be plated and cultured in vitro in its entirety, or as a further dissected micro-isolate that lacks the spiral limbus and spiral ganglion neurons. Using this method, primary explants can be maintained for 7-10 days. As an example of the utility of this procedure, organ of Corti explants will be electroporated with an exogenous DsRed reporter gene. This method provides an improvement over other published methods because it provides reproducible, unambiguous, and stepwise directions for the isolation, microdissection, and primary culture of the organ of Corti.

Primary Culture and Plasmid Electroporation of the Murine Organ of Corti.

This procedure describes a method for the isolation and culture of the murine organ of Corti with or without the spiral limbus and spiral ganglion neurons. We also demonstrate a method for the expression of an exogenous reporter gene in the organ of Corti explant by electroporation.

初代培養およびコルチのマウス臓器のプラスミドエレクトロ。

In all mammals, the sensory epithelium for audition is located along the spiraling organ of Corti that resides within the conch shaped cochlea of the ...

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 ...

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 ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

ショウジョウバエにおける温度嗜好挙動とその概日リズムの設計と解析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

大人のゼブラフィッシュの腎臓におけるネフロン構成と機能の解析

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Sequential Application of Glass Coverslips to Assess the Compressive Stiffness of the Mouse Lens: Strain and Morphometric Analyses

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the lens, leading to an increase in the lens&#39; optical power to focus on nearby objects, a process known as accommodation. Age-related changes in lens stiffness have been linked to presbyopia, a reduction in the lens&#39; ability to accommodate, and, by extension, the need for reading glasses. Even though mouse lenses do not accommodate or develop presbyopia, mouse models can provide an invaluable genetic tool for understanding lens pathologies, and the accelerated aging observed in mice enables the study of age-related changes in the lens. This protocol demonstrates a simple, precise, and cost-effective method for determining mouse lens stiffness, using glass coverslips to apply sequentially increasing compressive loads onto the lens. Representative data confirm that mouse lenses become stiffer with age, like human lenses. This method is highly reproducible and can potentially be scaled up to mechanically test lenses from larger animals.

Age-related increases in eye lens stiffness are linked to presbyopia. This protocol describes a simple, cost-effective method for measuring mouse lens stiffness. Mouse lenses, like human lenses, become stiffer with age. This method is precise and can be adapted for lenses from larger animals.

マウスレンズの圧縮剛性を評価するために、ガラスカバースリップの逐次適用：ひずみと形態計測分析

The eye lens is a transparent organ that refracts and focuses light to form a clear image on the retina. In humans, ciliary muscles contract to deform the ...

High-resolution Time-lapse Imaging and Automated Analysis of Microtubule Dynamics in Living Human Umbilical Vein Endothelial Cells

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes within individual endothelial cells (ECs). These processes are critical for homeostatic maintenance such as wound healing, and are also crucial in promoting tumor growth and metastasis. EC morphology is defined by the organization of the cytoskeleton, a tightly regulated system of actin and microtubule (MT) dynamics that is known to control EC branching, polarity and directional migration, essential components of angiogenesis. To study MT dynamics, we used high-resolution fluorescence microscopy coupled with computational image analysis of fluorescently-labeled MT plus-ends to investigate MT growth dynamics and the regulation of EC branching morphology and directional migration. Time-lapse imaging of living Human Umbilical Vein Endothelial Cells (HUVECs) was performed following transfection with fluorescently-labeled MT End Binding protein 3 (EB3) and Mitotic Centromere Associated Kinesin (MCAK)-specific cDNA constructs to evaluate effects on MT dynamics. PlusTipTracker software was used to track EB3-labeled MT plus ends in order to measure MT growth speeds and MT growth lifetimes in time-lapse images. This methodology allows for the study of MT dynamics and the identification of how localized regulation of MT dynamics within sub-cellular regions contributes to the angiogenic processes of EC branching and migration.

Protocols for Human Umbilical Vein Endothelial Cell (HUVEC) culture, transient transfection of fluorescently-labeled markers of microtubule growth, live-cell imaging and automated analysis of interphase microtubule growth dynamics are detailed.

リビングヒト臍帯静脈内皮細胞における高解像度タイムラプスイメージングと微小管ダイナミクスの自動解析

The physiological process by which new vasculature forms from existing vasculature requires specific signaling events that trigger morphological changes ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

「せん断リング」の組み立てと応用：小説内皮軌道、単方向および定期的な流動のためのモデルとせん断応力

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...