This work was funded in part by NIH R01 #DE021475 to CTM and an NSF Graduate Research Fellowship to NAE. Thanks to Miles Johnson for assistance with imaging and Priscilla Erickson for critical reading of the manuscript.

すべての魚の作業は、カリフォルニア大学バークレー校の施設内動物管理使用委員会（プロトコル番号R330）によって承認されました。安楽死は、0.1％炭酸水素ナトリウム39で緩衝0.025％トリカイン-Sへの浸漬を使用して行きました。全ての工程は室温で実施されます。 1.準備注意：しっかりと封止することができると水平に置くことが円錐管またはシンチレーションバイアルの手順1.1から1.5を実行します。魚は常に振とうする必要がありますが、静かに反転または染色液に魚のすべての側面を露出させ、汚れが均等に組織を貫通できるようにするために、チューブまたはバイアルのラックを振ることによってできるだけ頻繁に溶液を混合しないでください。液体の重い重量がシェーカーを破るように、プラットフォームシェーカー上で魚の大規模なバッチを置かないでください。 <ol><li> 10％中性緩衝ホルマリン（NBF）に一晩でエタノール中で保存したばかりの安楽死させた魚または魚のいずれかを修正しました。あるいは、4％のパラホルムを使用1×PBS溶液の代わりに10％NBF中のアルデヒド。 注：DNAを抽出した場合、エタノール中で固定し、店の前に尾や胸ひれの小さな部分をクリップ。 </li><li> 2時間（すなわち、pH約7.0である）、化学フード内で適切に修正を処分し、水道水で交換してください。それは多くの場合、酸性にすることができ、骨を脱灰することができるように脱イオン水を使用しないでください。 </li><li> 24時間水に1％KOHで0.008パーセントアリザリンレッドSで水や汚れの魚を削除します。魚が20mm未満の標準的な長さは、0.004パーセントアリザリンレッドSである（その後、希釈することができるアリザリンレッドSの100倍（0.8％）のストック溶液を作る）を使用します。 </li><li> （フード内の適切な廃棄容器に入れて）汚れを取り除き、数時間水道水に魚を置きます。必要に応じて水リンスはほとんど透明になるまで水を変更します。 </li><li>軽度の清算およびその後の解剖のために水を除去し、50％グリセロールに魚を置き、0.25％KOH。 注：この染色プロトコルは、前述の方法40,41から変更されています。 </li></ol><pクラス= &quot;jove_title&quot;&gt; 2。解剖注：関連するヘッド骨格形態の見直しについては、 図1を参照してください。 <img alt="図1" src="/files/ftp_upload/54056/54056fig1.jpg" /> 図1：トゲウオヘッド骨格形態アリザリンレッドは、ローダミンBフィルターセットの下で蛍光を用いて画像化イトヨヘッドを染色しました。有用な形態は標識されている：OP =主鰓蓋骨、Subop = subopercle、BSRの=鰓線、術前=前鰓蓋骨、Infraorb 1-3 =眼窩下1-3（またcircumorbitalsまたはsuborbitalsと呼ばれる）、デント=歯骨、Premax =前上顎骨、マックス=上顎、ナズ=鼻、緯度。 ethm =横篩骨、PSPH =副蝶形骨、フロン=前頭骨。トゲウオの頭部骨格のより詳細な説明については、アンカー（1974）31を参照してください。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54056/54056fig1large.jpg" target="_blank">の拡大版をご覧になるにはこちらをクリックしてください。</a>この図。 <ol><li>フラットフィッシュ（ 図2A）を置き、目を覆っている膜を穿刺するために〜45°の角度で、眼の側に鋭い＃5の時計メーカーの鉗子を挿入します。 </li><li>ピールヨーグルトのふた（ 図2B）を剥離に似た眼から離れた膜。 </li><li> 、目の後ろに開いた鉗子を挿入し、目の後ろの視神経のホールドをつかむと、目（ 図2C）を取り外します。それはメラニンをリークしますように目を開けたりしないでください。パンクした場合、メラニンは、後の工程の間に洗い流すことができます。 </li><li>反対側に繰り返します。 </li><li>後方から出発し、主鰓蓋骨フラップの下に一つの小さな切開鋏ブレードを置き、ドラッグ鋏ブレードは、背側主鰓蓋骨の上に、その後、眼窩（ 図2D）までの軟組織をカット。主鰓蓋骨の骨に背をカットします。 </li><li>前頭骨（アイソケットに背側）（ 図2E）をカットします。 </li><li>トンの周りの正中線副蝶形骨骨をカット眼窩（ 図2F）の彼が中心。 </li><li>反対側に主鰓蓋骨カットを繰り返します。 </li><li>主鰓蓋骨の下に鉗子を挿入し、ゆっくりと、まだ（ 図2G - H）結合した任意の軟組織をトリミングし、体から離し、顔の皮をむきます。鰓耙の最初の行を破壊しないように注意してください。 <ol><li>外装真皮主鰓蓋骨、前鰓蓋骨、subopercleを含む全体の舌骨の骨格を、そして、鉗子で、剥がしと前頭蓋顔面の骨格（歯骨、前上顎骨、および上顎を含む全体の顎を除去しながら正中basihyalから両側にceratohyalsを切り離します鰓線や根底にある背側と腹側の軟骨内の要素;および鼻、横篩骨洞、および眼窩下骨を含む頭蓋骨の前部には、 図1および図 2I）を参照してください。 </li><li>骨盤棘が体から出て折り畳むことができる、とホールドOをつかむために鉗子のハンドルとして機能することができますFが存在する場合。棘がロックされています。ロックを解除するには、静かに直接離れた魚体から鉗子で背骨を引っ張って、その後静かに魚に対して平ら背骨を押すように後方に曲がります。 </li></ol></li><li>挿入は離れて鰓スケルトン（ - K 図2J）に取り付けられ、残りの筋肉や靭帯をからかって、前方およびドラッグ鉗子（ちょうど腸管以下）鰓骨格に鉗子後部と腹側を閉じました。 </li><li>閉じた鉗子の先端を使用して、前方方向（ 図2L）に後方に腹braincaseに背鰓骨格を取り付ける筋肉を削り取ります。 </li><li>反対側に2.9と2.10を繰り返します。 </li><li>腸管のベースをつかみ、鰓骨格と腸管（図2M - N）を除去するために前方に引き出します。 </li><li>第五ceratobranchial（ 図2Oの最後に垂直なカット後部を行うことで、腸管を分離</strong&gt;）。 </li><li>鰓骨格の背側にbraincaseから残りの骨片を除去した後、背側歯プレートの二国間の組（ - D 図3A）との間に背側カット（後部に前方をカット）を作るために鰓バスケットにはさみを挿入します。カットが背側歯板の損傷を防ぐために、センタリングされていることを確認。 </li><li>鰓スケルトン（ 図3E）を開くを支援するために鰓スケルトン（腸管の前端）の後方端にゴム状の腸管内腔に2つの浅い横方向のカットを行います。 </li><li>魚や瓶にすべての組織片を置き、それ以上の清算が必要とされない場合は、穏やかなクリア、または100％のグリセロールを継続するために50％グリセロール、0.25％KOHでマイクロ遠心チューブに鰓骨格を配置します。彼らは追跡することができるように、一意の識別子とジャー、チューブにラベルを付けます。必要なクリア量が40メートルにわたって（主に魚、大きな大人の魚の大きさの関数であります標準的な長さのメートル）は、通常、追加の決済を必要とします。 </li></ol><img alt="図2" src="/files/ftp_upload/54056/54056fig2.jpg" /> 図2：トゲウオ鰓スケルトン解剖アリザリンレッドは、解剖の準備ができてイトヨの魚を染色しました。目は大規模なクリアから脱色されます。青い矢印は、動きの方向を示します。 （A）トゲウオヘッドの側面図、前部は右にあります。 （B）の目を覆っている膜の除去。 （C）目の除去。 （D）背側は、主鰓蓋骨の上にカット。 （E）前頭骨カット。 （F）副蝶形骨カット。 （G - I）顔面骨格の除去。腹鰓スケルトンの軟組織接続の（J）の除去。 （K - L）背鰓スケルトン接続の除去。 （&lt;強い&gt; M - N）鰓骨格の除去。 （O）鰓スケルトンから腸管を区切ります。詳細については、手順2.16を介して2.1を参照してください。スケールバー= 5ミリメートル。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54056/54056fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 3.鰓スケルトン再染色（必要な場合） <ol><li>より鰓スケルトン暗いまたは明確な組織を染色するために、50％グリセロール、0.25％KOH溶液を除去し、二回1％KOH（プラットフォームシェーカー上に水平に振とうしながら第二の24時間洗浄した1 5分間の洗浄）で洗浄しました。 </li><li> 24時間、1％KOHで0.008パーセントアリザリンレッドSで1％KOHおよび再汚れを取り除きます。 </li><li>汚れを削除し、24時間、1％KOHで交換してください。 </li><li> KOH溶液を除去し、50％グリセロール、0.25％KOHで交換してください。 </li></ol>鰓スケルトンを取り付け4. <ol><li>鰓骨格を削除します背側、22ミリメートル×60ミリメートルのガラスカバースリップの下の方に50％グリセロール、0.25％KOHまたは100％グリセロールと場所から（ 図3F）を上向き。鰓骨格の上に0.25％KOHまたは100％のグリセロール、50％グリセロールを数滴加えます。マイクロチューブに50％グリセロール、0.25％KOH〜100％グリセロール、変更溶液からの移行および&gt; 5分間の前に組織を平衡化するために実装に振る場合。 </li><li>スペーサーとして機能するようにカバースリップのどちらかの端に粘土と場所の二つの小さなボールをロールアウト。 </li><li>緩く前部鰓スケルトン（ 図3G）を平坦化するのに十分な圧力で上に第二のカバースリップを配置。 </li><li>ピールは、背側の歯のプレートを含む左背側フラップを開いて平らにし、カバースリップ（ 図3H）との間にスライド。 </li><li>右背側フラップ付き繰り返し技術とは、カバーガラス（ 図3I）の縁から離れて全体鰓骨格を押してください。 <ol><li>変更しますネイティブに、ピンセットでオープン両方の背側のフラップを保持し、慎重にカバースリップを置き、1滑らかな動きで鰓スケルトンを平坦化します。 </li><li>重力が鰓スケルトンがバックアップクローズすることはできませんので、別の方法として、アウト側方にそれぞれの背側を扇形に開く、1つのカバースリップ上で逆さま鰓骨格をマウントします。次に、2番目の22ミリメートルX 60ミリメートルガラスカバースリップでカバーし、準備を反転。 注：異なる実装技術は、個々のために良いか悪いか仕事する傾向があります。それぞれを試してみて、最も快適に感じるものを参照してください。 </li></ol></li><li>軽く鰓骨格を維持するのに十分粘土ボールを平らにするため、トップカバースリップ上に平らに取り付けられたキーを押しますが、試料を粉砕しないように注意してください。 <ol><li>実装工程中に、ceratobranchialsが回転し、耙の行を曖昧にすることができます。カバースリップして再配向ceratobranchialsまたは全体鰓スケルトンの間に鉗子をスライドさせることによってこの問題を解決。 </li></ol></li><li>店はROでスライドトレイに平らプレップオム温度。 100％グリセロール中にマウントされ、調製物は、少なくとも十年ブリッジカバースリップの間保存することができます。イソプロパノールまたはエタノールとカバーのヒントとクリーン鉗子とはさみ。 </li></ol><img alt="図3" src="/files/ftp_upload/54056/54056fig3.jpg" /> 図3：鰓スケルトンのフラット鰓骨格を取り付け操作と実装が示されています。青い矢印は、動きの方向を示します。 （A）鰓スケルトン背側を上に。 （B - D）背側の歯のプレート間の回転と切開。さらに腸管のベースを開くには、軟組織中の（E）横カット。 （F）取り付けるための準備ができてカバーガラスの下の方に置かれて鰓スケルトン。 （G）（背側歯プレート上）鰓骨格の前半に配置された第2のカバースリップ。 （H - I）背歯プレートのフラップを開けると2つのカバースリップの間にスライドさせることによって鰓骨格のフラットマウント。詳細については、4.6まで手順4.1を参照してください。スケールバー= 5ミリメートル。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/54056/54056fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a>

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The authors have nothing to disclose.

The branchial skeleton is a complex set of bones in the throat of a fish that manipulates, filters, and masticates food items on their way to the esophagus. Many interesting trophic traits including the patterning of gill rakers, pharyngeal teeth, and branchial bones vary across and within species. The majority of these traits are difficult to near impossible to accurately measure with the branchial skeleton in situ (e.g., gill raker length, branchial bone length). This flat-mounting protocol places all...

多様性の信じられないほどの量は、特に魚類の中で、脊椎動物のうち、頭部骨格に存在します。多くの場合、この多様性は、異なる給餌戦略1を容易に- 4を 、そして外部と内部の両方の頭蓋顔面パターン形成に大きな変更を伴うことができます。鰓スケルトンは魚の喉の内部に位置し、口腔の大部分を取り囲みます。鰓骨格は鰓をサポート前部4そのうちの5連続的相同セグメントで構成されています。一緒にこれらの5つのセグメントは、魚や彼らの食糧5との間のインタフェースとして機能します。鰓耙、咽頭歯、および鰓骨などの形質の多数の変動は、食品のさまざまな種類の効率的な採餌に貢献しています。 トゲウオは、北半球全体の淡水湖や小川植民地化先祖の海洋形後の適応放散を受けました。食生活の変化海の小さな動物プランクトンから淡水でより大きな獲物には、いくつかの頭蓋顔面の特性6の劇的な栄養変化をもたらしました。多くの研究は、トゲウオ7で外部頭蓋顔面の違いに焦点を当ててきたが- 13、重要な頭蓋顔面の変更は内部鰓スケルトンで繰り返し進化します。形態学的に異なるトゲウオ集団との間の肥沃なハイブリッドを作成する機能は、鰓スケルトンへの進化の変化の遺伝的基礎をマッピングするための絶好の機会を提供します。 生態学的意義の一つ栄養特性は、鰓耙、鰓骨の前部と後部の顔を並べると獲物のアイテムをフィルタリングするために使用されている定期的な皮膚、骨のパターニングです。一般的に小さな獲物のアイテムを餌に魚がより長く、より密に間隔を置い鰓が大きい獲物14,15を餌に魚に比べ耙持っている傾向があります。鰓耙の変化が報告されている両方のwithinと種間14-19、および鰓放蕩者のパターニングの側面は、栄養ニッチとフィットネス16に貢献しています。数十年にわたる研究が盛んthreespineトゲウオで17鰓放蕩者の数と長さの変化を報告している- 21;しかし、これらの研究は、一般的に鰓耙の最初の行に焦点を当てます。最近の研究は、1または単一の鰓の放蕩者が理解するために行以上のことを学ぶことの重要性を強調23、長さ24間隔鰓スケルトン22,23を横断し、鰓放蕩者で単一の行全体で鰓放蕩者数の遺伝的制御にモジュール性を示しています鰓放蕩者の減少の発生遺伝的基盤。 生態系と生物医学の両方の重要性の第二の栄養特性は、咽頭歯のパターニングです。魚類における歯は咽頭歯として知られている、両方の口腔顎にし、鰓スケルトンに配置することができます。口腔歯は、pのために主に使用されています27 -咽頭歯は咀嚼と獲物操作25のために使用されている間レイキャプチャします。両方のセットは、共有発達機構を介して形成し、相同28発達と考えられています。このようなゼブラフィッシュ、不足口腔および背側咽頭歯29のようないくつかの種は、他の種は、複数の歯付きceratobranchialsを持ってpharyngobranchials、時にはbasihyal歯と30を hypobranchialsながら、それによって興味深いモジュール性が発生します。トゲウオでは、咽頭歯は前部と後部31を pharyngobranchials上の第五ceratobranchialと背に腹側に発見されました。トゲウオ供給上のキネマティクスは、口腔顎は主に獲物捕獲および咽頭顎に咀嚼を残して吸引送り9を容易にするために使用されることを示します。シクリッドでは、下咽頭顎形態は劇的32,33が変化し、適応および栄養ニッチ34と相関することが示されています。マルチPLE淡水トゲウオ集団は腹側咽頭歯数23,35,36の劇的な増加を進化させてきました。最近の研究は、この進化した歯利得の発達の遺伝的基礎は淡水トゲウオ36の2つの独立して誘導された集団における大部分は別個のものであることを示しました。哺乳動物の歯とは違って、魚は成人期37を通じて継続的に自分の歯を再生成します。これらの前述の高い歯付淡水集団の両方が再生36の遺伝的基礎を研究するまれな脊椎動物のシステムを提供すること、加速歯の交換レートを進化させてきました。 淡水トゲウオで繰り返し進化してきた第三の栄養特性は長く、それぞれ38 epibranchialとceratobranchial骨、上下の顎の鰓弓の分節同族体、です。長い鰓の骨が大きく口腔を与え、おそらくより大きな獲物アイテムはcできるようにするために適応されていますonsumed。また、他の魚で、epibranchial骨は背側咽頭歯プレート25のうつ病のために重要です。鰓耙と咽頭歯と同じように、鰓骨は、内部および容易に視覚化または定量化することは、困難です。 ここでは、分析する詳細なプロトコルを提示し、重要な頭蓋顔面形質の様々な簡単に可視化および定量化を可能にする、鰓骨格をフラットマウント。このプロトコルは、トゲウオの解剖を説明しているが、これと同じ方法は、他の魚の様々に取り組んでいます。

<table><tbody><tr><td>Potassium Hydroxide (KOH)</td><td>EMD</td><td>PX1480-1</td><td></td></tr><tr><td>Glycerol</td><td>Sigma-Aldrich</td><td>G7893-4L</td><td></td></tr><tr><td>10% Neutral Buffered Formalin (NBF)</td><td>Azer Scientific</td><td>NBF-4-G</td><td></td></tr><tr><td>Alizarin Red S</td><td>EMD</td><td>AX0485-3</td><td></td></tr><tr><td>Microscope Cover Glasses 22 mm x 60 mm</td><td>VWR</td><td>16004-350</td><td></td></tr><tr><td>100 mm x 10 mm Glass Petri Dish</td><td>Kimble Chase</td><td>23064-10010</td><td>To dissect samples on</td></tr><tr><td>Sylgard 184 Silicone Elastomer Kit</td><td>Ellsworth Adhesives</td><td>184 SIL ELAST KIT 0.5KG</td><td>Can be poured into glass or plastic Petri dishes to make dissecting plates</td></tr><tr><td>Modeling Clay</td><td>Sargent Art</td><td>22-4000</td><td>1 lb cream</td></tr><tr><td>Scintillation Vials (case of 500)</td><td>Wheaton</td><td>986586</td><td>Borosilicate Glass with Screw Cap</td></tr><tr><td>Forceps-Dumont #5 Inox (Biologie tip)</td><td>FST</td><td>11252-20</td><td>Dumostars are an alternative</td></tr><tr><td>Dissecting Scissors&amp;nbsp;</td><td>FST</td><td>15003-08</td><td>Alternate sizes are available depending on size of sample</td></tr><tr><td>Dissecting Microscope</td><td>Leica</td><td>S6E with KL300 LED</td><td>Many other models work nicely, having a flat base helps</td></tr><tr><td>Microcentrifuge Tubes 1.7 ml</td><td>Denville</td><td>C2170</td><td></td></tr><tr><td>Cardboard slide tray</td><td>Fisher</td><td>12-587-10</td><td></td></tr></tbody></table>

dissection and flat-mounting of the threespine stickleback branchial skeleton

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology of the fish branchial skeleton is matched to a species' diet. Threespine stickleback fish (Gasterosteus aculeatus) have emerged as a model system to study the genetic and developmental basis of evolved differences in a variety of traits. Marine populations of sticklebacks have repeatedly colonized countless new freshwater lakes and creeks. Adaptation to the new diet in these freshwater environments likely underlies a series of craniofacial changes that have evolved repeatedly in independently derived freshwater populations. These include three major patterning changes to the branchial skeleton: reductions in the number and length of gill raker bones, increases in pharyngeal tooth number, and increased branchial bone lengths. Here we describe a detailed protocol to dissect and flat-mount the internal branchial skeleton in threespine stickleback fish. Dissection of the entire three-dimensional branchial skeleton and mounting it flat into a largely two-dimensional prep allows for the easy visualization and quantification of branchial skeleton morphology. This dissection method is inexpensive, fast, relatively easy, and applicable to a wide variety of fish species. In sticklebacks, this efficient method allows the quantification of skeletal morphology in genetic crosses to map genomic regions controlling craniofacial patterning.

<html>重要な栄養形質の様々な定量化することができる解剖し、フラットマウントさ鰓骨格にこのプロトコルの結果（ 図4）。背側から見ると、鰓耙、すべての咽頭歯板、およびほぼすべての鰓骨のすべての行を簡単に可視化し、22定量することができる- 24,35,36,38,42を 。アリザリンレッドSはまた、他のマーカー（ 例えば 、?...

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Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species.

解剖やイトヨ鰓スケルトンのフラットマウント

The posterior pharyngeal segments of the vertebrate head give rise to the branchial skeleton, the primary site of food processing in fish. The morphology ...

dissection-flat-mounting-threespine-stickleback-branchial

Universidad de Cartagena UDEC

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Biology

9.7K ビュー.  University of California, Berkeley. The branchial skeleton, including gill rakers, pharyngeal teeth, and branchial bones, serves as the primary site of food processing in most fish. Here we describe a protocol to dissect and flat-mount this internal skeleton in threespine sticklebacks. This method is also applicable to a variety of other fish species. 

ビデオ: Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

蛍光タンパク質の発現において<em&gt; Branchiostoma lanceolatum</em&gt;未受精卵へのmRNA注入による

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

発生生物学

Microinjection for Transgenesis and Genome Editing in Threespine Sticklebacks

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and behavioral phenotypes. The remarkably diverse phenotypes that have evolved as marine populations adapt to countless freshwater environments, combined with the ability to cross marine and freshwater forms, provide a rare vertebrate system in which genetics can be used to map genomic regions controlling evolved traits. Excellent genomic resources are now available, facilitating molecular genetic dissection of evolved changes. While mapping experiments generate lists of interesting candidate genes, functional genetic manipulations are required to test the roles of these genes. Gene regulation can be studied with transgenic reporter plasmids and BACs integrated into the genome using the Tol2 transposase system. Functions of specific candidate genes and cis-regulatory elements can be assessed by inducing targeted mutations with TALEN and CRISPR/Cas9 genome editing reagents. All methods require introducing nucleic acids into fertilized one-cell stickleback embryos, a task made challenging by the thick chorion of stickleback embryos and the relatively small and thin blastomere. Here, a detailed protocol for microinjection of nucleic acids into stickleback embryos is described for transgenic and genome editing applications to study gene expression and function, as well as techniques to assess the success of transgenesis and recover stable lines.

Transgenic manipulations and genome editing are critical for functionally testing the roles of genes and cis-regulatory elements. Here a detailed microinjection protocol for the generation of genomic modifications (including Tol2-mediated fluorescent reporter transgene constructs, TALENs, and CRISPRs) is presented for the emergent model fish, the threespine stickleback.

Threespineトゲウオにおける遺伝子導入およびゲノム編集のためのマイクロインジェクション

The threespine stickleback fish has emerged as a powerful system to study the genetic basis of a wide variety of morphological, physiological, and ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

ゼブラフィッシュジョーバイオメカニクスを調査するために有限要素モデルの構築

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

Characterization Of Multi-layered Fish Scales (Atractosteus spatula) Using Nanoindentation, X-ray CT, FTIR, and SEM

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle shells provides unique design principles with potentials for guiding the design of protective materials and systems in the future. Understanding the structure-property relationships for these material systems at the microscale and nanoscale where failure initiates is essential. Currently, experimental techniques such as nanoindentation, X-ray CT, and SEM provide researchers with a way to correlate the mechanical behavior with hierarchical microstructures of these material systems1-6. However, a well-defined standard procedure for specimen preparation of mineralized biomaterials is not currently available. In this study, the methods for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of A. spatula using nanoindentation, FTIR, SEM, with energy-dispersive X-ray (EDX) microanalysis, and X-ray CT are presented.

Characterization Of Multi-layered Fish Scales Atractosteus spatula Using Nanoindentation, X-ray CT, FTIR, and SEM

This paper presents the methods used for probing spatially correlated chemical, structural, and mechanical properties of the multilayered scale of Atractosteus spatula (A. spatula) using nanoindentation, Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and X-ray computed tomography (X-ray CT). The experimental results have been used to investigate the design principles of protective biological materials.

多層魚の鱗の特性（<em&gt; Atractosteusヘラ</em&gt;）ナノインデンテーション、X線CT、FTIR、およびSEMを用いて

The hierarchical architecture of protective biological materials such as mineralized fish scales, gastropod shells, ram&#8217;s horn, antlers, and turtle ...

生物工学

Sequential Photo-bleaching to Delineate Single Schwann Cells at the Neuromuscular Junction

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system and has served as guiding model to study synaptic structure and function. In mouse NMJs motor axon terminals form pretzel-like contact sites with muscle fibers. The motor axon and its terminal are sheathed by SCs. Over the past decades, several transgenic mice have been generated to visualize motor neurons and SCs, for example Thy1-XFP1 and Plp-GFP mice2, respectively.
Along motor axons, myelinating axonal SCs are arranged in non-overlapping internodes, separated by nodes of Ranvier, to enable saltatory action potential propagation. In contrast, terminal SCs at the synapse are specialized glial cells, which monitor and promote neurotransmission, digest debris and guide regenerating axons. NMJs are tightly covered by up to half a dozen non-myelinating terminal SCs - these, however, cannot be individually resolved by light microscopy, as they are in direct membrane contact3.
Several approaches exist to individually visualize terminal SCs. None of these are flawless, though. For instance, dye filling, where single cells are impaled with a dye-filled microelectrode, requires destroying a labelled cell before filling a second one. This is not compatible with subsequent time-lapse recordings3. Multi-spectral "Brainbow" labeling of SCs has been achieved by using combinatorial expression of fluorescent proteins4. However, this technique requires combining several transgenes and is limited by the expression pattern of the promoters used. In the future, expression of "photo-switchable" proteins in SCs might be yet another alternative5. Here we present sequential photo-bleaching, where single cells are bleached, and their image obtained by subtraction. We believe that this approach - due to its ease and versatility - represents a lasting addition to the neuroscientist's technology palette, especially as it can be used in vivo and transferred to others cell types, anatomical sites or species6.
In the following protocol, we detail the application of sequential bleaching and subsequent confocal time-lapse microscopy to terminal SCs in triangularis sterni muscle explants. This thin, superficial and easily dissected nerve-muscle preparation7,8 has proven useful for studies of NMJ development, physiology and pathology9. Finally, we explain how the triangularis sterni muscle is prepared after fixation to perform correlated high-resolution confocal imaging, immunohistochemistry or ultrastructural examinations.

Visualizing individual cells in densely packed tissues, such as terminal Schwann cells (SCs) at neuromuscular junctions (NMJs), is challenging. "Sequential photo-bleaching" allows delineating single terminal SCs, for instance in the triangularis sterni muscle explant, a convenient nerve-muscle preparation, where sequential bleaching can be combined with time-lapse imaging and post-hoc immunostainings.

シーケンシャル神経筋接合部におけるシングルシュワン細胞の輪郭を描くのにフォトブリーチング

Sequential photo-bleaching provides a non-invasive way to label individual SCs at the NMJ. The NMJ is the largest synapse of the mammalian nervous system ...

神経科学

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

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

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

解剖やゼブラフィッシュ胚の横方向の取付：脊髄発達の解析

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

A 3-D Tail Explant Culture to Study Vertebrate Segmentation in Zebrafish

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from the presomitic mesoderm (PSM) as the tail end of the embryo elongates posteriorly. Somites form with regular periodicity and scale in size. Zebrafish is a popular model organism as it is genetically tractable and has transparent embryos that allow for live imaging. Nevertheless, during somitogenesis, fish embryos are wrapped around a large, rounding yolk. This geometry limits live imaging of PSM tissue in zebrafish embryos, particularly at higher resolutions that require a close objective working distance. Here, we present a flattened 3-D tissue culture method for live imaging of zebrafish tail explants. Tail explants mimic intact embryos by displaying a proportional slowdown of axis elongation and shortening of rostrocaudal somite lengths. We are further able to stall axis elongation speed through explant culture. This, for the first time, enables us to untangle the chemical input of signaling gradients from the mechanistic input of axial elongation. In future studies, this method can be combined with a microfluidic setup to allow time-controlled pharmaceutical perturbations or screening of vertebrate segmentation without any drug penetration concerns.

Here, we present the protocol for 3-D tissue culture of the zebrafish posterior body axis, enabling live study of vertebrate segmentation. This explant model provides control over axis elongation, alteration of morphogen sources, and subcellular resolution tissue-level live imaging.

ゼブラフィッシュにおける脊椎動物のセグメンテーションを研究する3D尾翼植物培養

Vertebrate embryos pattern their major body axis as repetitive somites, the precursors of vertebrae, muscle, and skin. Somites progressively segment from ...

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

脊椎動物の軸の伸びとセグメンテーションを研究するための3次元および4次元の可視化と解析アプローチ

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

Stab Wound Injury Model of the Adult Optic Tectum Using Zebrafish and Medaka for the Comparative Analysis of Regenerative Capacity

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury model was developed in the adult optic tectum of zebrafish and medaka and comparative histological and molecular analyses were performed to elucidate the molecular mechanisms regulating the high regenerative capacity of this tissue across these fish species. Here a stab wound injury model is presented for the adult optic tectum using a needle and histological analyses for proliferation and differentiation of the neural stem cells (NSCs). A needle was manually inserted into the central region of the optic tectum, and then the fish were intracardially perfused, and their brains were dissected. These tissues were then cryosectioned and evaluated using immunostaining against the appropriate NSC proliferation and differentiation markers. This tectum injury model provides robust and reproducible results in both zebrafish and medaka, allowing for comparing NSC responses after injury. This method is available for small teleosts, including zebrafish, medaka, and African killifish, and enables us to compare their regenerative capacity and investigate unique molecular mechanisms.

A mechanical brain injury model in the adult zebrafish is described to investigate the molecular mechanisms regulating their high regenerative capacity. The method explains to create a stab wound injury in the optic tectum of multiple species of small fish to evaluate the regenerative responses using fluorescent immunostaining.

ゼブラフィッシュとメダカを用いた成体視神経筋の刺創損傷モデルによる再生能力の比較解析

While zebrafish have a superior capacity to regenerate their central nervous system (CNS), medaka has a lower CNS regenerative capacity. A brain injury ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

ホールマウントによるゼブラフィッシュ胚標本ステンドの観察および分析のためのフラットマウントの準備<em&gt;その場で</em&gt;ハイブリダイゼーション

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

解剖やイトヨ鰓スケルトンのフラットマウント

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蛍光タンパク質の発現において