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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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://www-jove-com.remotexs.ntu.edu.sg/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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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ancient+gene+network+is+co-opted+for+teeth+on+old+and+new+jaws.">An ancient gene network is co-opted for teeth on old and new jaws.</a> PLoS Biol. 7 (2), e1000031 (2009).</li><li>Stock, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+dentition+in+comparative+context.">Zebrafish dentition in comparative context.</a> J. Exp. Zool. B. Mol. Dev. Evol. 308, 523-549 (2007).</li><li>Liem, K., Greenwood, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+approach+to+the+phylogeny+of+the+pharyngognath+teleosts.">A functional approach to the phylogeny of the pharyngognath teleosts.</a> Am. Zool. 21 (1), 83-101 (1981).</li><li>Anker, G. 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B. 281, (2014).</li><li>Leary, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> AVMA Guidelines for the Euthanasia of Animals. , (2013).</li><li>Bell, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+phenetics+and+genetics.">Evolutionary phenetics and genetics.</a> Evol. Genet. Fishes. , 431-528 (1984).</li><li>Taylor, W. R., Van Dyke, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Revised+procedures+for+staining+and+clearing+small+fishes+and+other+vertebrates+for+bone+and+cartilage+study.">Revised procedures for staining and clearing small fishes and other vertebrates for bone and cartilage study.</a> Cybium. 9 (2), 107-119 (1985).</li><li>Erickson, P. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+190+base+pair,+TGF-&#946;+responsive+tooth+and+fin+enhancer+is+required+for+stickleback+Bmp6+expression.">A 190 base pair, TGF-&#946; responsive tooth and fin enhancer is required for stickleback Bmp6 expression.</a> Dev. Biol. 401 (2), 310-323 (2015).</li><li>Miller, C. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cis-Regulatory+changes+in+Kit+ligand+expression+and+parallel+evolution+of+pigmentation+in+sticklebacks+and+humans.">cis-Regulatory changes in Kit ligand expression and parallel evolution of pigmentation in sticklebacks and humans.</a> Cell. 131 (6), 1179-1189 (2007).</li><li>Aigler, S. R., Jandzik, D., Hatta, K., Uesugi, K., Stock, D. 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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 branchial skeletal elements in a simple two-dimensional plane for ease of imaging and quantification of multiple traits. 
One limitation of this protocol is that although teeth, gill rakers, and most endochondral bones within this flat-mounted two-dimensional prep can be easily imaged and quantified, the second through fourth epibranchials, that have much more three-dimensional shapes, are not easily visualized and would require additional dissection to accurately measure. A second limitation of this protocol is the method is minimally destructive to the fish head. However, only two bones are damaged with a single cut each, the parasphenoid and the frontal bone. All other craniofacial skeletal elements can be returned to and measured at later points in time.
This protocol, although developed for sticklebacks, can be used in many other fish species, although sometimes modifications are necessary. For example, some fish species like zebrafish have a non-ossified dorsal chewing pad44 requiring the dorsal cut to be lateral, around this plate. The posterior end of the fifth ceratobranchial is curved is some species (e.g., zebrafish and other Cypriniformes) and must be trimmed or much larger clay spacers used to allow flat mounting. Particularly large specimens can be mounted between two pieces of glass or acrylic in the same manner. Species with a particularly thick epithelial layer covering tooth plates can be restained and cleared after dissection or more aggressively cleared with 2% KOH. Fish in this situation may also be presoaked in 1 or 2% KOH prior to Alizarin staining to increase dye penetrance. 
An alternative more technologically advanced method for imaging the branchial skeleton is Micro Computed Tomography (&#181;CT)23,45. However, &#181;CT requires an expensive scanner, is much more time consuming, and requires considerable training. Instead, this dissection protocol is inexpensive, quick, and amenable to high throughput studies required for quantitative studies such as quantitative trait loci (QTL) mapping genomic regions controlling evolved changes in craniofacial patterning. Additionally, these dissections can be performed at any larval or adult time point22,35,36,38 to study skeletal development. Although fish head anatomy is complex, this simple protocol allows easy visualization of the internal branchial skeleton, a critically important component of trophic morphology.

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>Sodium Hydroxide (KOH)</td>
			<td>EMD</td>
			<td>PX1480-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Sigma-Alderich</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>116-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Cover Glasses 22x60mm</td>
			<td>VWR</td>
			<td>16004-350</td>
			<td></td>
		</tr>
		<tr>
			<td>100x10mm 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>1lb cream</td>
		</tr>
		<tr>
			<td>Scintillation Vials (case of 500)</td>
			<td>Wheaton</td>
			<td>66021-668</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&#160;</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 1.7mL</td>
			<td>Denville</td>
			<td>C-2170</td>
			<td></td>
		</tr>
		<tr>
			<td>Cardboard slide tray</td>
			<td>Fisher</td>
			<td>12-587-10</td>
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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 、トランスジェニックGFP 42）との二重標識し、可視化する別の方法を可能ローダミンまたは類似の赤色フィルタに蛍光を発します。蛍光イメージングまたは表現型が計画されている場合、蛍光が暗闇の中で店舗プレップので、光の中ですぐにフェードインします。腹側から見ると、鰓を可視化し、それらの色素沈着は、43を定量化することができます。プレップは年間100％グリセロール中で保存することができます。 4.JPG &quot;/&gt; 図4：代表トゲウオ鰓骨格染色し、クリア鰓骨格の2つの例が示されています。 （A）赤ラベルされた骨を示す明視野像。 （B）ローダミンBフィルターセットの下で蛍光画像。耙、歯、および骨の例は、それぞれ、キャレット、矢じり、およびアスタリスクで標識されています。スケールバー= 2ミリメートル。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54056/54056fig4large.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>

Watch this Scientific Journal Video about Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton at JoVE.com

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

Nanyang Technological University

Research

JoVE Journal

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

Dissection of Drosophila Ovaries

ショウジョウバエの卵巣の解剖

生物学

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

ショウジョウバエ幼虫NMJの解剖

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

ロブスターの胃の肉眼解剖、ロブスター属Americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

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

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

成体マウスから海馬歯状回の解剖

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

Dissection of Organs from the Adult Zebrafish

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

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

アダルトゼブラフィッシュからの臓器の解剖

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

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

の解剖と染色ショウジョウバエ幼虫卵巣

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

Dissection of the Adult Zebrafish Kidney

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

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

アダルトゼブラフィッシュ腎臓の解剖

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

Dissection and Mounting of Drosophila Pupal Eye Discs

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes. It contains approximately 800 separate eye units, termed ommatidia1. Each ommatidium contains eight neuronal photoreceptors that develop from undifferentiated cells following the passage of the morphogenetic furrow in the third larval instar2. Following the sequential differentiation of the photoreceptors, non-neuronal cells develop, including cone and pigment cells, along with mechanosensory bristle cells3. Final differentiation processes, including the structured arrangement of all the ommatidial cell types, programmed cell death of undifferentiated cell types and rhodopsin expression, occurs through the pupal phase4-7. This technique focuses on manipulating the pupal eye disc, providing insight and instruction on how to dissect the eye disc during the pupal phase, which is inherently more difficult to perform than the commonly dissected third instar eye disc. This technique also provides details on immunostaining to allow the visualization of various proteins and other cell components.

Dissection and Mounting of Drosophila Pupal Eye Discs

The goal of this technique is to enable researchers to perform dissection, immunostaining and mounting of pupal eye discs from Drosophila melanogaster of any age.

解剖と実装<em&gt;ショウジョウバエ</em&gt;蛹アイディスク

The Drosophila melanogaster eye disc is a powerful system that can be used to study many different biological processes.  It contains approximately 800 ...

Dissection of the Auditory Bulla in Postnatal Mice: Isolation of the Middle Ear Bones and Histological Analysis

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called the auditory bulla houses the ossicles, whereas the auditory capsule encloses the inner ear, namely the cochlea and semicircular canals. Murine ossicles are essential for hearing and thus of great interest to researchers in the field of otolaryngology, but their metabolism, development, and evolution are highly relevant to other fields. Altered bone metabolism can affect hearing function in adult mice, and various gene-deficient mice show changes in morphogenesis of auditory ossicles in utero. Although murine auditory ossicles are tiny, their manipulation is feasible if one understands their anatomical orientation and 3D structure. Here, we describe how to dissect the auditory bulla and capsule of postnatal mice and then isolate individual ossicles by removing part of the bulla. We also discuss how to embed the bulla and capsule in different orientations to generate paraffin or frozen sections suitable for preparation of longitudinal, horizontal, or frontal sections of the malleus. Finally, we enumerate anatomical differences between mouse and human auditory ossicles. These methods would be useful in analyzing pathological, developmental and evolutionary aspects of auditory ossicles and the middle ear in mice.

We present a protocol to isolate the auditory bulla, capsule, and ossicles from postnatal mice for whole mount and histological analysis.

生後マウスにおける聴覚ブラの解剖：中耳の骨の単離および組織学的解析

In most mammals, auditory ossicles in the middle ear, including the malleus, incus and stapes, are the smallest bones. In mice, a bony structure called ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

郭清と霊長類の顔の進化における DiceCT 明らかにする別の方法で非表示のデータを逆に

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...