Name: dissection and flat-mounting of the threespine stickleback branchial skeleton
Uploaded: 2016-05-07T15:00:00.000+00:00
Duration: 8 min 2 s
Description: Watch this Scientific Journal Video about Dissection and Flat-mounting of the Threespine Stickleback Branchial Skeleton at JoVE.com

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.

Tüm balık çalışmaları California-Berkeley Üniversitesi Kurumsal Hayvan Bakım ve Kullanım Komitesi (protokol numarası R330) tarafından onaylandı. Ötenazi% 0.1 sodyum bikarbonat 39 ile tamponlanmış% 0.025 Tricaine-S daldırma kullanılarak gerçekleştirilmiştir. Bütün aşamalar oda sıcaklığında gerçekleştirilir. 1. Hazırlık Not: sıkıca kapatabilir ve yatay döşenebilir konik tüpler veya sintilasyon şişeleri adımları 1.1-1.5 gerçekleştirin. Balıklar sürekli sarsılmış olması gerekir, fakat nazikçe tersini veya boyama çözeltisine balık her tarafını ortaya çıkarmak ve leke eşit dokuya nüfuz sağlamak için tüpler veya şişelere raf sallayarak mümkün olduğunca sık çözüm karıştırmak kalkmayın. sıvı ağır çalkalayıcı kıracak gibi bir çalkalama platformunda balık büyük bir toplu koymayın. <ol><li> gece boyunca,% 10 nötral tamponlu formalin (NBF), etanol içinde depolanmış ya da taze ötanazi balık ya da balık düzeltildi. Alternatif olarak,% 4 paraformaldehid kullanımı1x PBS çözeltisi yerine% 10 NBF aldehit. Not: DNA ayıklanması ise, etanol içinde fiksasyon ve saklamak için önce kuyruk veya göğüs yüzgeçleri küçük bir bölümünü klibi. </li><li> 2 saat boyunca (yani ~ pH 7.0) kimyasal bir kaput düzgün düzeltmenin atınız ve musluk suyu ile değiştirin. genellikle, asidik olabilir ve kemik kirecini gibi de-iyonize su kullanmayın. </li><li> 24 saat boyunca su içinde% 1 KOH% 0.008 Alizarin Kırmızısı S su ve leke balık çıkarın. Balık az 20 mm standart uzunluk için% 0.004 Alizarin Kırmızı S. (daha sonra seyreltilmiş olabilir Alizarin Kırmızı S 100x (% 0.8) stok solüsyonu olun) kullanın. </li><li> Bir kaç saat musluk suyunda leke (başlık uygun çöp bidonuna koyarak) ve yer balık çıkarın. su ile durulama çoğunlukla netleşene kadar gerektiği gibi suyu değiştirin. </li><li> % 50 gliserol, hafif takas ve sonraki diseksiyon için% 0.25 KOH içine su ve yer balık çıkarın. Not: Bu boyama protokolü daha önce tarif edilen yöntemler 40,41 modifiye edilir. </li></ol><pclass = &quot;jove_title&quot;&gt; 2. teşrih Not: İlgili kafa iskelet morfolojisi bir inceleme için Şekil 1&#39;e bakınız. <img alt="Şekil 1" src="/files/ftp_upload/54056/54056fig1.jpg" /> Şekil 1:. Stickleback kafa iskelet morfolojisi Alizarin Kırmızı bir rodamin B filtre kümesi altında floresan ile görüntülenmiş threespine dikenli kafasını boyandı. Faydalı morfolojisi etiketlenmiştir: Op = opercle, Subop = suboperkülün, BSRs = branchiostegal ışınları, Ameliyat öncesi = preopercle, Infraorb 1-3 = infraorbital 1-3 (diğer adıyla circumorbitals veya suborbitals), Dent = dentary, PREMAX = premaxilla, Max = maksilla Nas = burun, Lat. ethm = yanal etmoid, Psph = parasphenoid, Fron = frontal kemik. Dikenli kafa iskeletinin daha ayrıntılı bilgi için (1974) 31 Anker bkz. <a href="https://www.jove.com/files/ftp_upload/54056/54056fig1large.jpg" target="_blank">Büyük halini görmek için tıklayınız</a>bu figür. <ol><li> Balık düz (Şekil 2A) Lay ve göz kapsayan membran delinme için ~ 45 ° &#39;lik açıyla gözün yan içine keskin 5. saatçilik forseps yerleştirin. </li><li> Uzak bir yoğurt kapağı (Şekil 2B) soyma benzer göz, gelen membran soyun. </li><li> Gözün arkasındaki açık forseps yerleştirin gözün arkasındaki optik sinirin tutunmasını ve göz (Şekil 2C) çıkarın. o melanin sızıntısı gibi göz delmeyin. delinmiş ise, melanin sonraki adımlarda sırasında uzak yıkanabilir. </li><li> diğer tarafta tekrarlayın. </li><li> Posterior itibaren opercle flep altında küçük bir diseksiyon makas bıçak yerleştirin sürükleme makas bıçak dorsal opercle üstünde, daha sonra göz soket (Şekil 2B) aracılığıyla yumuşak dokuyu kesti. opercle kemiğe dorsal kesin. </li><li> Frontal kemik (göz soket dorsal) (Şekil 2E) kesin. </li><li> t etrafında orta hat parasphenoid kemik kesmekgöz çukurları (Şekil 2F) o merkezi. </li><li> Rakip tarafta tekrarlayın opercle kesti. </li><li> Opercle altında forseps yerleştirin ve yavaşça hala (Şekil 2G - H) bağlı herhangi bir yumuşak doku kırparak, vücuttan uzak yüzünü soymak. solungaç diken ilk satırı bozmak için özen gösterin. <ol><li> Dış dermal opercle, preopercle, suboperkülün dahil olmak üzere tüm hyoid iskeleti ve; forseps ile, uzak soyulması ve ön kraniofasyal iskelet (dentary, premaxilla ve maksilla dahil olmak üzere tüm çene çıkarırken orta hat basihyal gelen her iki tarafta ceratohyals ayırmak branchiostegal ışınları ve altta yatan dorsal ve ventral endochondral elemanları ve burun, yan etmoid dahil kafatasının ön kısmı ve infraorbital kemikler,) Şekil 1 ve 2I bakın. </li><li> Pelvik dikenler vücuttan dışarı katlanabilir ve forseps tutun o kapmak için bir tutamak olarak hizmet verebilirne zaman f mevcut. Dikenler yerine kilitleyin. Kilidi açmak için, yavaşça doğrudan uzak balık vücuttan forseps ile omurga çekin, sonra yavaşça balık karşı düz omurga basın posterior viraj. </li></ol></li><li> Ekle dışında brankiyal iskeletinin (- K Şekil 2J) bağlı kalan kasları ve bağları alay, öne ve sürükle forseps (sadece gut tüp altında) brankiyal iskeletine forseps posterior ve ventral kapattı. </li><li> Anterior yönde (Şekil 2L) bir posterior ventral braincase dorsal brankiyal iskeleti bağlama kasları kazınması, kapalı forseps ipuçlarını kullanarak. </li><li> Karşı tarafın 2.9&#39;a ve 2.10 tekrarlayın. </li><li> Bağırsak tüp tabanını kavrayın ve brankiyal iskelet ve bağırsak tüp (Şekil 2M - N) çıkarmak için öne doğru çekin. </li><li> (Beşinci ceratobranchial sonuna Şekil 2O dik kesme posterior yaparak bağırsak tüpü ayırın </strong&gt;). </li><li> Brankiyal iskeletin sırt tarafında braincase kalan kemik parçaları çıkardıktan sonra, dorsal diş plakalarının ikili kümeleri arasında dorsal kesim (posterior anterior kesme) (- D Şekil 3A) yapmak için brankiyal sepetin içine makas yerleştirin. Emin olun kesme dorsal diş plakaları zarar vermemek için ortalanır. </li><li> Brankiyal iskeleti (Şekil 3E) açılması ile yardımcı olmak için brankiyal iskeletin (bağırsak borusunun ön ucu) arka ucundaki lastik bağırsak lümeninde iki sığ yan keser. </li><li> balık ve bir kavanoza tüm doku parçaları yerleştirin ve başka temizleme gerekli ise yumuşak temizleme, ya da% 100 gliserol devam% 50 gliserol,% 0.25 KOH içeren bir mikrosantrifüj tüpü içine brankiyal iskelet yerleştirin. benzersiz bir tanımlayıcı ile etiket kavanoz ve tüpler böylece izlenebilir. Gerekli temizleme miktarı 40 m üzerinde (çoğunlukla balık, büyük bir yetişkin balık büyüklüğünün bir fonksiyonudurstandart boyda m), tipik olarak ek takas gerektirir. </li></ol><img alt="şekil 2" src="/files/ftp_upload/54056/54056fig2.jpg" /> Şekil 2:. Stickleback brankiyal iskelet diseksiyonu Alizarin Kırmızı diseksiyon için threespine dikenli balık hazır boyandı. Göz geniş açıklıkta gelen depigmente edilir. Mavi oklar hareket yönünü gösterir. (A), dikenli kafasının yanal görünüşüdür, ön sağ etmektir. (B) göz kaplayan zarın çıkarılması. Gözün (C) çıkarılması. (D) dorsal opercle üzerinde kesti. (E) Cephe kemik kesim. (F) Parasphenoid kesti. (G - I) yüz iskeletinin çıkarılması. Ventral brankiyal iskelet yumuşak doku bağlantılarının (J) Sökme. (K - L) dorsal brankiyal iskelet bağlantılarının kaldırılması. (&lt;strong&gt; M - N) brankiyal iskeletinin çıkarılması. (O) brankiyal iskeletinden bağırsak tüpü ayırın. Daha fazla bilgi için adımları 2.16 ile 2.1 bakınız. Ölçek çubuğu = 5 mm. <a href="https://www.jove.com/files/ftp_upload/54056/54056fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> 3. Brankial İskelet Yeniden boyama (Gerekli ise) <ol><li> solungaç iskelet koyu ya da açık dokuya daha fazla leke için,% 50 gliserol,% 0.25 KOH çözeltisi çıkarın ve iki kez% 1 KOH (bir çalkalama platformunda yatay çalkalama sırasında, ikinci bir 24 saat yıkama, ardından bir ila beş dakikalık bir yıkama) ile yıkayın. </li><li> 24 saat boyunca% 1 KOH içinde 0.008% Alizarin Kırmızı S% 1 KOH ve yeniden leke çıkarın. </li><li> leke çıkarın ve 24 saat süreyle% 1 KOH ile değiştirin. </li><li> KOH çözeltisi çıkarın ve% 50 gliserol,% 0.25 KOH ile değiştirin. </li></ol> 4. Brankial Skeleton Montaj <ol><li> Brankial iskeleti kaldırsırt tarafında bir 22 mm x 60 mm cam kapak slip altına yakın% 50 gliserol,% 0.25 KOH veya% 100 gliserol ve yerden (Şekil 3F) yukarı bakacak. branşiyal iskeletin üstüne% 50 gliserol,% 0.25 KOH veya% 100 gliserol birkaç damla ekleyin. Bir mikrosantrifüj tüp içinde% 50 gliserol,% 0.25 KOH% 100 gliserol, değişim çözümden geçiş ve&gt; 5 dakika boyunca önceden doku dengelenmesi montaj sallamak durumunda. </li><li> ayırıcılar olarak hareket etmek kapak kayma her iki ucunda kil modelleme ve yerin iki küçük topları dışarı rulo. </li><li> Gevşek ön brankiyal iskeleti (Şekil 3G) düzleştirmek için yeterli basınçla üstünde ikinci bir lamel yerleştirin. </li><li> Peel, dorsal diş plakalar dahil sol dorsal kapağını açmak düzleştirmek ve lamelleri (Şekil 3H) arasında slayt. </li><li> Sağ dorsal flep ile tekrarlayın tekniği ve lamel (Şekil 3H) kenarından uzak tüm brankiyal iskeleti itin. <ol><li> değiştirmekdoğal, forseps ile açık hem dorsal flep tutun ve dikkatli bir şekilde tek bir hareketle brankiyal iskelet düzleşme, üzerine lamel yerleştirin. </li><li> yerçekimi brankiyal iskelet geri kapatmak için izin vermez Alternatif olarak, dışarı yanal her dorsal yüzü eğimlendirme, bir kapak kayma ters brankiyal iskeleti monte edin. Sonra ikinci 22 mm x 60 mm cam kapak slip ile kaplayın ve hazırlık ters. Not: Farklı montaj teknikleri iyi ya da kötü her birey için çalışmak eğilimindedir. Her deneyin ve en rahat hissettiği bakın. </li></ol></li><li> Hafif brankiyal iskeleti tutmak için yeterli kil topları düzleştirmek için üst lamel düz monte edilmiş basın, ancak örnek ezmek için özen gösterin. <ol><li> Montaj işlemi sırasında, ceratobranchials döndürmek ve diken bir satır gizleyebilir. lamelleri ve yeniden yönlendirme ceratobranchials veya tüm brankiyal iskelet arasındaki forseps kaydırarak bu sorunu. </li></ol></li><li> Mağaza ro slayt tepsilerde düz prepsom sıcaklığı. % 100 gliserol monte, prepler en az on yıl için köprülü lamelleri arasında saklanabilir. Temiz forseps ve izopropanol veya etanol ve kapak ipuçları makas. </li></ol><img alt="Şekil 3," src="/files/ftp_upload/54056/54056fig3.jpg" /> Şekil 3:. Daire brankiyal iskelet Manipülasyon montaj ve brankiyal iskeletin montajı gösterilmiştir. Mavi oklar hareket yönünü gösterir. (A) Brankial iskelet dorsal yüzü yukarı. Dorsal diş plakaları arasında - (B D) Rotasyon ve kesi. Yumuşak dokuda (E) Yanal kesim daha bağırsak borusunun tabanını açmak için. Montaj için hazır bir lamel altına yerleştirilen (F) Brankial iskelet. (Dorsal diş plakaları üzerinde) brankiyal iskeletin ön yarısında yerleştirilen (G) İkinci lamel. (H - I)dorsal diş plaka kanatlarını açarak ve iki kapak fişleri arasında kaydırarak brankiyal iskeletin düz montaj. Daha fazla bilgi için adımları 4,6 ile 4.1 bakınız. Ölçek çubuğu = 5 mm. <a href="https://www.jove.com/files/ftp_upload/54056/54056fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

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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...

çeşitlilik inanılmaz bir miktar özellikle balıklar arasında, omurgalılar arasında baş iskeleti var. Birçok durumda bu çeşitlilik, farklı beslenme stratejileri 1 kolaylaştırır - 4, ve her ikisi de dış ve iç kraniofasyal desenlendirme büyük değişiklikler içerebilir. brankiyal iskelet bir balık boğaz dahili olarak yer alan ve ağız boşluğu en çevreler. brankiyal iskelet solungaçları destekleyen ön dördü 5 seri homolog segmentleri, oluşmaktadır. Birlikte bu beş segment balık ve onların yiyecek 5 arasında bir arayüz olarak işlev görür. solungaç diken, yutak dişleri ve brankiyal kemikleri de dahil olmak üzere özellikleri çok sayıda varyasyon yiyecek farklı türlerde etkili yiyecek arama katkıda bulunur. Sticklebacks kuzey yarımkürede boyunca tatlı su gölleri ve dereleri kolonize atalarının okyanus formları sonra bir adaptif radyasyon uğramıştır. diyet kaymasıtatlı daha büyük av için okyanusta küçük zooplankton birkaç kraniofasyal özellikleri 6 dramatik trofik değişmesine neden olmuştur. Birçok çalışma Sticklebacks 7 dış kraniofasial farklılıkları odaklanmış olsa da - 13, önemli kraniofasial değişiklikler iç brankiyal iskelette defalarca gelişmeye. Morfolojik farklı dikenli popülasyonlar arasındaki verimli melezler oluşturma yeteneği brankiyal iskeletine evrimleşmiş değişikliklerin genetik temelini eşleştirmek için mükemmel bir fırsat sağlar. ekolojik öneme sahip biri tropik özelliği solungaç diken, brankiyal kemiklerin anterior ve posterior yüzleri satır ve av öğeleri filtrelemek için kullanılan periyodik dermal kemiklerin desenleme olduğunu. Genellikle küçük av öğeleri beslemek balıklar daha uzun ve daha yoğun aralıklı solungaç büyük yırtıcı 14,15 beslenen balıklara oranla diken sahip olma eğilimindedir. solungaç diken varyasyon bildirilmiştir hem within ve türler 14-19 arasında ve solungaç havalandırma makinesi desenlendirme yönleri trofik nişler ve fitness 16 katkıda bulunur. Araştırma Yıllar yoğun threespine Sticklebacks 17 solungaç raker numarası ve uzunluk varyasyonu belgeledi - 21; Ancak, bu çalışmalar genellikle solungaç diken ilk satırında odaklanmak. Son çalışmalar, bir veya bir tek solungaç havalandırma makinesi anlamak için satır daha çalışmanın önemini vurgulayarak 23 ve uzunluk 24 aralık brankiyal iskelet 22,23 genelinde ve solungaç Tarak tek satır boyunca solungaç havalandırma makinesi sayısının genetik kontrol modüler göstermiştir solungaç raker azaltma gelişimsel genetik temeli. hem ekolojik ve biyomedikal önemi ikinci trofik özellik faringeal dişlerin desenleme olduğunu. Balıklarda Diş yutak dişleri olarak da bilinir, hem sözlü çene ve brankiyal iskeletinde yer olabilir. Ağız diş p amacıyla kullanılır27 - yutak dişleri çiğneme ve av manipülasyon 25 için kullanılır ise rey yakalamak. Her iki takım ortak gelişimsel mekanizmaları aracılığıyla oluşturur ve homolog 28 gelişimsel olarak kabul edilir. Bazen böyle zebrafish, eksikliği ağız ve dorsal faringeal dişler 29 diğer türler birden dişli ceratobranchials, pharyngobranchials varken gibi türler, ve basihyal dişli ve 30 hypobranchials sayede ilginç modülerlik oluşur. Sticklebacks olarak, yutak dişleri ön beşinci ceratobranchial ve dorsal üzerinde ventral bulundu ve posterior 31 pharyngobranchials edilir. Dikenli beslenme ile ilgili Kinematik sözlü çene öncelikle av yakalama ve yutak çenesine çiğneme bırakarak emme besleme 9 kolaylaştırılması kullanılır gösteriyor. Çiklitlerinde, alt yutak çene morfolojisi dramatik 32,33 değişir ve adaptif ve trofik niş 34 ile korelasyon olduğu gösterilmiştir. Çokple tatlısu dikenli popülasyonları ventral faringeal diş sayısı 23,35,36 dramatik artışlar meydana gelmiştir. Son çalışmalar bu evrimleşmiş diş kazanç gelişimsel genetik temeli tatlısu Sticklebacks 36 iki bağımsız toplumlarında büyük ölçüde farklı olduğunu ortaya koymuştur. Memeli dişlerin aksine, balık yetişkin yaşam 37 boyunca sürekli dişlerini yeniden. Bu daha önce açıklanan yüksek dişli tatlı su popülasyonlarının ikisi de rejenerasyon 36 genetik temelini incelemek için nadir bir omurgalı sistemi sağlayarak, hızlandırılmış protez oranını gelişmiştir. Tatlısu Sticklebacks defalarca gelişti üçüncü trofik özellik artık sırasıyla 38 epibranchial ve ceratobranchial kemikler, üst ve alt çene solungaç kemeri segmental homologları vardır. Uzun brankiyal kemikleri daha geniş bir yanak boşluğu görüşmek ve muhtemelen daha büyük av öğeleri c olmak için izin verdiği için uyarlanabilironsumed. Ayrıca, diğer balıklarda, epibranchial kemikler dorsal faringeal diş plakaları 25 depresyon açısından önemlidir. solungaç diken ve yutak dişleri gibi, brankiyal kemikler kolayca görselleştirmek veya ölçmek zordur, bu nedenle iç ve vardır. Burada incelemek için detaylı bir protokol mevcut ve önemli kraniofasyal özelliklerin çeşitli kolay görselleştirme ve sayılmasına olanak tanıyan, brankiyal iskeleti düz monte edin. Bu protokol, dikenli diseksiyon açıklanmış olsa da, aynı yöntem, diğer balıkların çeşitli çalışır.

<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> Önemli trofik özelliklerin çeşitli ölçülebilir bir disseke ve düz monte brankiyal iskelet Bu protokol sonuçları (Şekil 4). Dorsal görünümünde, solungaç diken, tüm faringeal diş plakaları, ve neredeyse tüm brankiyal kemiklerin tüm satırları kolayca görüntülendi ve 22 sayısal olabilir - 24,35,36,38,42. Alizarin Kırmızı S diğer belirteçleri (örneğin, transgenik GFP 42) ve görselle?...

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.

Diseksiyon ve Threespine Stickleback Brankial İskelet Düz montaj

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

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Biology

9.7K Görüntüleme.  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. 

Video: 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.

Floresan protein ifadesindeki<em&gt; Branchiostoma lanceolatum</em&gt; Döllenmemiş Oositlerinin içine mRNA Enjeksiyon

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

Gelişim Biyolojisi

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 Sticklebacks içinde Transgenesis ve Genom Kurgu Mikroenjeksiyon

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.

Zebra balığı Çeneli Biyomekanik Araştırma Sonlu Elemanlar Modelleri Bina

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.

Çok katmanlı Balık Ölçekler (Of Karakterizasyonu<em&gt; Atractosteus spatula</em&gt;) Nanoindentation, X-ray CT, FTIR ve SEM kullanma

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

Biyomühendislik

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.

Sıralı Nöromusküler Kavşağı&#39;nda Tek Schwann Hücreleri belirginleştiren Photo-beyazlatma

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 ...

Nörobilim

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.

Diseksiyon ve Zebra balığı Embriyolar Yanal Montaj: Omurilik Kalkınma Analizi

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.

Zebra Balıklarında Omurgalı Segmentasyonunu İncelemek için 3-D Kuyruk Eksplant Kültürü

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.

Omurgalı Eksenel Uzama ve Segmentasyonunu İncelemek için Üç ve Dört Boyutlu Görselleştirme ve Analiz Yaklaşımları

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.

Rejeneratif Kapasitenin Karşılaştırmalı Analizi için Zebra Balığı ve Medaka Kullanan Yetişkin Optik Tektumunun Bıçak Yarası Yaralanma Modeli

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.

Tüm Dağı ile Zebra balığı Embriyo örnekler Vitray gözlenmesi ve Analiz Daire Dağı Hazırlık<em&gt; In situ</em&gt; Melezleme

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

Diseksiyon ve Threespine Stickleback Brankial İskelet Düz montaj

Özet

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Floresan protein ifadesindeki

Threespine Sticklebacks içinde Transgenesis ve Genom Kurgu Mikroenjeksiyon

Zebra balığı Çeneli Biyomekanik Araştırma Sonlu Elemanlar Modelleri Bina

Çok katmanlı Balık Ölçekler (Of Karakterizasyonu

Sıralı Nöromusküler Kavşağı'nda Tek Schwann Hücreleri belirginleştiren Photo-beyazlatma

Diseksiyon ve Zebra balığı Embriyolar Yanal Montaj: Omurilik Kalkınma Analizi

Zebra Balıklarında Omurgalı Segmentasyonunu İncelemek için 3-D Kuyruk Eksplant Kültürü

Omurgalı Eksenel Uzama ve Segmentasyonunu İncelemek için Üç ve Dört Boyutlu Görselleştirme ve Analiz Yaklaşımları

Rejeneratif Kapasitenin Karşılaştırmalı Analizi için Zebra Balığı ve Medaka Kullanan Yetişkin Optik Tektumunun Bıçak Yarası Yaralanma Modeli

Tüm Dağı ile Zebra balığı Embriyo örnekler Vitray gözlenmesi ve Analiz Daire Dağı Hazırlık