この作品は、パーデュー大学で生物科学科からのスタートアップ資金によってサポートされています。

パート1：マイクロダイセクション前の準備<ol><li>ソリューション<ol class="lalpha"><li> E3培地4（5 mMのNaCl、0.17 mMの塩化カリウム、0.33 mMのCaCl 2および0.33 mMのMgSO 4を ）。 </li><li>リンゲル溶液1、5は （116 mMのNaCl、2.9 mMの塩化カリウム、1.8 mMのCaCl 2を 、5mMのHEPES、pH7.2）、フィルタは滅菌。 </li><li> 5N NaOHで。 </li></ol></li><li>タングステン針の化学エッチング<ol class="lalpha"><li>粘土でペトリ皿に5N NaOHを含む小さなビーカーを固定します。 </li><li>それは、NaOH溶液と接触するように、ビーカーの側面にペーパークリップを取り付けます。 </li><li>ペーパークリップやタングステン線の短い断片（〜2センチメートル）の一端に正極へのDC電源から負極を接続します。 </li><li>小さな粘土のボールを持つタングステン線のもう一方の端をラップし、NaOH溶液にこの端を浸し。 </li><li>約3Vまで電圧を増加し、良好なエッチング速度が得られるまでNaOH溶液で上下線を浸漬。良い品質の針は通常10分でエッチングすることができる。 </li><li>ソリューションにさらされるタングステン線は徐々に薄くなるでしょう。粘土のボールがオフに低下すると、まだ正極に接続されているワイヤの反対側には、鋭い針の形状を持つことになります。 </li><li>塩の付着物を除去するためにリンゲル液またはE3に鋭い針を洗浄します。 </li><li>テープや取り扱いを容易にするためニードルホルダーが木製のアプリケーターに針を取り付けます。 </li></ol></li><li>培養プレート<ol class="lalpha"><li>ファルコン60 × 15mmのポリスチレン培養プレートは、網膜のマイクロダイセクションのために使用する準備が整いました。 </li><li> RPE -添付の網膜を切開するため、30分解剖する前に表面の接着性を取り除くためにリンゲル液を含有する培養プレートで完全にtwo胚をつぶす。まもなく解剖する前に、新鮮なリンゲル液で広範囲にプレートを洗浄。 </li></ol></li><li>胚のコレクションとステージング<ol class="lalpha"><li>大人のゼブラフィッシュは、4、5を説明するように維持されます。 </li><li>分周器によって繁殖の前に夜をペアワイズ静的な繁殖タンク内の親魚を区切ります。 </li><li>部屋のライトが次の朝にオンになって後に分周器を取り外します。両親は10分間隔で30分おきに交差することができます。各交差点の後に魚を区切ります。 </li><li> 28で別々にE3培地に各交差点から収集された胚を維持℃に</li><li>確立された基準（1）（6）によれば、特定の時点で10〜12時間後に受精（HPF）でと再演するすぐに解剖する前段階の胚、。 </li></ol></li></ol>パート2：解剖-脳の除去と眼のばく露1 <ul><li>このセクションで説明する手順は、網膜（パート3A）及びRPE -添付の網膜を（パート3B）解剖のための共通です。 </li><li>すべての微細な解剖はデュモン鉗子の先端でおこなわれており、組織を位置決めするためのデュモン鉗子の別のペアによって援助されています。必要に応じて化学的にエッチングされたタングステンの針は、細かい操作に使用することができます。 </li><li>すべての微細な解剖は1X目的または同等の装備オリンパスSZX16顕微鏡下で〜5倍速の倍率で実行されます。 </li><li> 28でベンチトップインキュベータにE3培地で胚を維持℃の解剖時に簡単に胚のアクセスのための顕微鏡の隣にある。 </li><li>顕微鏡のステージは、遺伝子発現に及ぼす温度変動の影響を最小限に抑えるためにサーモプレートで28℃に加温している。 </li></ul><ol><li>第1部＃3で説明したように調製されたファルコン60 × 15mmの培養プレートでリンゲル液に特定の発達段階にある胚を移す。 </li><li>体内から速やかに前方トランクの部分で頭を切り落とします。 </li><li>支援鉗子で培養プレートにこの組織の後端を固定。 </li><li>前額から始まる背側に頭を開きます。 </li><li>目の内側が露出され、さらなる操作のために上向きになるように脳を取り外します。 </li></ol>パート3a：網膜切開1 <ol><li>として第2部で説明されている胚を準備する。 </li><li>網膜に小さな開口部が見られるまで慎重に鉗子の先端が上向きに直面している眼球の内側に露出したRPEを磨きます。 </li><li>ブラッシングと網膜の内側はほぼすべてが露出するまでアクションを剥離を継続する。露出網膜を傷つけないし、パンチは避けてください。 </li><li>今下向き直面している網膜、アプローチに横方向のRPEを削除することと培養プレートの表面から約45 °の角度でブラシ。 </li><li> cにRPEのデタッチ部分を固定することによって、鋸状縁に比較的しっかりと接続されてRPEを削除します。ulture郭清のためのプレート、そして次にゆっくりと網膜を持ち上げます。 </li><li>残留RPEをクリーンアップするために培養皿の底に網膜をロールバックします。 <ol class="lalpha"><li>我々は、胚が粉砕されると、この特定のファルコンのポリスチレン培養プレートは、約20分間RPEの優先的な密着性を持っていることに気づいた。このプロパティは、RPEの残党の除去のために利用されている。しかし、網膜細胞は、高倍率下で密接に検査することができるより少ない程度に表面に棒を行います。一つは、RPEの除去と網膜の整合性の完全性とのバランスを持っています。 </li><li>レンズは、しばしば培養プレートに付着し、圧延工程中に網膜から切り離します。時折、それはレンズの表面に渦巻くアクションでエッチングされたタングステン針でレンズを取り外すときに必要です。 </li></ol></li><li>これらの手順は、正常組織の整合性を損なうことなく、網膜からRPEを削除することができます。これは、良好な全体的な形態（図1B及びB&#39;）と組織学（図1B&quot;）で示される、矢印特に、光受容体層とRPEとの間の細胞外マトリックスの保全が（図1B非常に良いです。&quot;;全胚（図1A&quot;））から、組織型で比較する。 </li></ol>一部3B：RPE接続網膜解剖3 <ol><li>として第2部で説明されている胚を準備する。 </li><li>助手の鉗子によって培養プレートに頭部を固定。後部側面からそっと目を持ち上げて、前方側にロールバックします。 <ol class="lalpha"><li> RPE層の外側に推定脈絡膜と強膜組織は、皮膚に、比較的しっかりと接続されており、主に前方側に注意深く目を圧延して剥離することができる。 </li></ol></li><li>目の側面が露出していると眼がまだ肌に接続されているときに後のタングステン針でレンズを外します。 </li><li>これらのプロシージャは、正常網膜で全体RPE層を保持することができます。推定脈絡膜と強膜組織は、主に（図1C、C&#39;とC&quot;）は削除することができます。 </li></ol>パート4：RNAの作業のための組織サンプルの収集<ol><li>解剖のサンプルは1日より前に説明したように下流のRNAの特性評価のためのRNaseフリーのマイクロチューブにTRIZOLに集めることができます。 </li></ol>代表的な結果<img src="/files/ftp_upload/2028/2028fig1.jpg" alt="図1" /> 図1（）解剖前の52 HPFにおけるゼブラフィッシュ幼生の頭部の横方向と（&#39;）背ビュー。幼虫の頭の中で（）＆（&#39;）の（&quot;）に対応する組織切片（B）側面と（B&#39;）54 HPFにおける解剖網膜の背ビュー。網膜の表面には、両方の側面から無傷だったと背側ビュー。（B）＆（B&#39;）の解剖網膜の（B&quot;）に対応する組織切片。網膜と網膜積層構造は無傷であった。特に、光受容体層とRPE（間細胞外マトリックスは、&quot;（、矢印、矢印）も解剖網膜B）に保存されていました&quot;。 （C）側方および52 HPFにおける解剖RPEに接続された網膜の（C&#39;）内側のビュー。 RPE層はまた、切除した組織の組織切片（C&quot;）で示された、無傷と連続だった。C&#39;の白い領域は、視神経（矢印）である。組織は、組織試料を4％で収集し、修正された3を説明するようにパラホルムアルデヒド。埋め込 ​​み、これらの試料のセクショニングのプラスチックを行った。スケールバー=50μmのは。

<ol>
	<li>Leung, Y. F., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+zebrafish+embryonic+retina.">Gene expression profiling of zebrafish embryonic retina.</a> Zebrafish. 2, 269-283 (2005).</li><li>Leung, Y. F., Ma, P., Link, B. A., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factorial+microarray+analysis+of+zebrafish+retinal+development.">Factorial microarray analysis of zebrafish retinal development.</a> Proc Natl Acad Sci U S A. 105, 12909-12914 (2008).</li><li>Leung, Y. F., Ma, P., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+zebrafish+embryonic+retinal+pigment+epithelium+in+vivo.">Gene expression profiling of zebrafish embryonic retinal pigment epithelium in vivo.</a> Invest Ophthalmol Vis Sci. 48, 881-890 (2007).</li><li>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=.">.</a> Zebrafish : a practical approach. , (2002).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+book+:+a+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">The zebrafish book : a guide for the laboratory use of zebrafish (Danio rerio).</a> , (2000).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li><li>Fadool, J. M., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+model+system+for+the+study+of+eye+genetics.">Zebrafish: a model system for the study of eye genetics.</a> Prog Retin Eye Res. 27, 89-110 (2008).</li></ol>

動物実験は、パーデュー大学の動物実験委員会が定めるガイドラインおよび規則に従って行った。

ゼブラフィッシュの眼の組織の顕微解剖は、効果的に無傷の網膜とRPEに接続された網膜を取得することができます。これは実質的に特定の眼組織（すなわち、網膜や網膜色素上皮）に関連する発現研究を支援します。実際に、我々が正常に全体の網膜1とRPE 3のRNAの発現プロファイルを取得するには、次の手順を利用してきた。これらのプロファイルのユーティリティが強く網...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cordless pestle motor</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>47747-370</td>
<td>&nbsp;</td>
<tr>
<td>DC power supply</td>
<td>&nbsp;</td>
<td>Lascar</td>
<td>PSU130</td>
<td>Any DC supply would work. The specific voltage of a different machine will need further optimization.</td>
</tr>
<tr>
<td>Disposable pestle &amp; microtube, 1.5 mL (DNase, RNase and pyrogen-free)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>47747-366</td>
<td>These are used for tissue collection in TRIzol for expression analysis.</td>
</tr>
<tr>
<td>Dumont #5 forceps, Tips: 0.05 x 0.01mm, Inox</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>500341</td>
<td>Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.</td>
</tr>
<tr>
<td>Dumont #5SF forceps, Tips: 0.025 x 0.005mm, Inox</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-00</td>
<td>Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.</td>
</tr>
<tr>
<td>Falcon polystyrene culture plates, 60 X 15 mm</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>351007</td>
<td>These plates are used as dissection plates.</td>
</tr>
<tr>
<td>Olympus SZX16 Stereomicroscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>SZX16</td>
<td>Any stereomicroscope would work. We used Leica stereomicroscope in previous studies1-3 without any issues. We also use the 1X objective exclusively for the dissection even though we have a 2X objective installed. </td>
</tr>
<tr>
<td>Sharpening stone</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>29008-01</td>
<td>Use this to sharpen the tip of the forceps if necessary</td>
</tr>
<tr>
<td>Thermo plate</td>
<td>&nbsp;</td>
<td>Tokai Hit</td>
<td>MATS-U55SZX2B</td>
<td>This is used to maintain the temperature of the tissue throughout dissection and minimize the influence of temperature fluctuation on gene expression. We also put the whole microscope in an environmentally controlled room at 28&deg;C during dissection in previous studies1-3 with good success.</td>
</tr>
<tr>
<td>Trizol, 100 mL</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15596-026</td>
<td>&nbsp;</td>
<tr>
<td>tungsten wire, 0.015 inch diameter</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TGW1510</td>
<td>&nbsp;</td>
<tr>
<td>Wooden Applicator</td>
<td>&nbsp;</td>
<td>Puritan</td>
<td>807</td>
<td>This is used for holding the chemically-etched tungsten needle.</td>
</tr>		</tbody>
		</table>
		</div>

microdissection of zebrafish embryonic eye tissues

ゼブラフィッシュは、その急速の眼の発生に関する研究のための人気の動物モデルである<em&gt; EX子宮</em&gt;開発と良い産卵。 3日後の受精（DPF）によって、幼虫は、最初の視覚的な応答が表示されます。多くの遺伝子は、適切な眼の発生を制御するために同定されたが、我々は、基礎となる遺伝的構造の完全な理解にはほど遠いですされています。全ゲノムの遺伝子発現プロファイリングは、眼の開発のための遺伝子調節ネットワークを解明するために有用なツールです。しかし、ゼブラフィッシュの胚の眼の小さいサイズは、それが挑戦的な発現解析のための完全な、純粋な眼の組織を得ることができる。レンズの径が100μm以下であるたとえば、2日目と3の間に目の前後の長さは、唯一の約200から300μmである。また、網膜の根底にある網膜色素上皮（RPE）はちょうど単層上皮である。遺伝子発現プロファイルは全胚から得られる一方で、彼らは正確にこれらの組織の表現を表すものではありません。したがって、純粋な組織は、眼の開発の成功した遺伝子発現プロファイリングのために取得する必要があります。この問題に対処するために、我々は、眼の形態形成の主要な段階をカバーする1から3 DPF、から添付RPEで無傷の網膜と網膜を顕微解剖する手法を開発しました。すべての手順は、標準的な実体顕微鏡下で微細な鉗子と一般的な実験室の電源で行うことができます。網膜の解剖のために、単層RPEは、アクションと解剖のための培養プレートの表面にRPEの残党の優先的な遵守をブラッシングして除去し、剥離​​する。 RPEが完全に網膜で保存できるように、RPEに接続された網膜節郭清については、解剖のプレートにRPEの遵守は、解剖前に削除されます。この組織を慎重に持ち上げアクションは、効率的に推定脈絡膜と強膜を分離することができます。レンズは、化学的にエッチングされたタングステンの針が両方のケースで削除することができます。要するに、我々のアプローチは、そのまま眼の組織を得ることができますし、正常にゼブラフィッシュの網膜の組織特異的発現プロファイルを研究するために利用されている<sup&gt; 1、2</sup&gt;と網膜色素上皮<sup&gt; 3</sup&gt;。

Watch this Scientific Journal Video about Microdissection of Zebrafish Embryonic Eye Tissues at JoVE.com

Microdissection of Zebrafish Embryonic Eye Tissues

この記事では、1〜3日postfertilizationの胚から、接続されている網膜色素上皮がある場合とない場合のゼブラフィッシュ網膜を顕微解剖するアプローチを説明します。

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 ...

microdissection-of-zebrafish-embryonic-eye-tissues

Research

JoVE Journal

Biology

16.0K ビュー.  Purdue University. この記事では、1〜3日postfertilizationの胚から、接続されている網膜色素上皮がある場合とない場合のゼブラフィッシュ網膜を顕微解剖するアプローチを説明します。

ビデオ: Microdissection of Zebrafish Embryonic Eye Tissues

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

哺乳動物の組織のレーザーキャプチャーマイクロダイセクション

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

生物学

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

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

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

共焦点顕微鏡によるゼブラフィッシュ胚の脳のイメージングを生きる

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

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

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 can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

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 ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

組織標的胚キメラ：ゼブラフィッシュの原腸胚細胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

遺伝子発現解析のための胚ゼブラフィッシュとcDNA合成からのRNA分離

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

ブラックウィドウスパイダーシルクを生産腺の顕微解剖

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

ディテールに秘められたゼブラフィッシュ胚心臓の免疫染色

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

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 ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

のためのシステム<em&gt; ex vivoで</em胚膵臓&gt;培養

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

ゼブラフィッシュ胚骨格筋のレーザー招いた傷害

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...