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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/54056/54056fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

<ol>
	<li>Cooper, W. J., Westneat, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Form+and+function+of+damselfish+skulls:+rapid+and+repeated+evolution+into+a+limited+number+of+trophic+niches.">Form and function of damselfish skulls: rapid and repeated evolution into a limited number of trophic niches.</a> BMC Evol. Biol. 9 (24), (2009).</li><li>Albertson, R. C., Kocher, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+developmental+basis+of+cichlid+trophic+diversity.">Genetic and developmental basis of cichlid trophic diversity.</a> Heredity. 97 (3), 211-221 (2006).</li><li>Martin, C. H., Wainwright, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+novelty+is+linked+to+exceptional+rates+of+morphological+diversification+in+two+adaptive+radiations+of+cyprinodon+pupfish.">Trophic novelty is linked to exceptional rates of morphological diversification in two adaptive radiations of cyprinodon pupfish.</a> Evolution. 65 (8), 2197-2212 (2011).</li><li>Wainwright, P. C., 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=The+evolution+of+pharyngognathy:+A+phylogenetic+and+functional+appraisal+of+the+pharyngeal+jaw+key+innovation+in+labroid+fishes+and+beyond.">The evolution of pharyngognathy: A phylogenetic and functional appraisal of the pharyngeal jaw key innovation in labroid fishes and beyond.</a> Syst. Biol. 61 (6), 1001-1027 (2012).</li><li>Sibbing, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+capture+and+oral+processing.">Food capture and oral processing.</a> Cyprinid Fishes. , 377-412 (1991).</li><li>Bell, M., Foster, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Evolutionary Biology of the Threespine Stickleback. , (1994).</li><li>Kimmel, C. B., 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=Evolution+and+development+of+facial+bone+morphology+in+threespine+sticklebacks.">Evolution and development of facial bone morphology in threespine sticklebacks.</a> Proc. Natl. Acad. Sci. U. S. A. 102 (16), 5791-5796 (2005).</li><li>Mcgee, M. D., Wainwright, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+evolution+as+a+generator+of+phenotypic+diversity+in+threespine+stickleback.">Convergent evolution as a generator of phenotypic diversity in threespine stickleback.</a> Evolution. 67 (4), 1204-1208 (2013).</li><li>McGee, M. D., Schluter, D., Wainwright, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+basis+of+ecological+divergence+in+sympatric+stickleback.">Functional basis of ecological divergence in sympatric stickleback.</a> BMC Evol. Biol. 13, 277 (2013).</li><li>McGuigan, K., Nishimura, N., Currey, M., Hurwit, D., Cresko, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+genetic+variation+in+static+allometry+in+the+threespine+stickleback.">Quantitative genetic variation in static allometry in the threespine stickleback.</a> Integr. Comp. Biol. 50 (6), 1067-1080 (2010).</li><li>Caldecutt, W. J., Bell, M. A., Buckland-Nicks, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexual+dimorphism+and+geographic+variation+in+dentition+of+threespine+stickleback,+Gasterosteus+aculeatus.">Sexual dimorphism and geographic variation in dentition of threespine stickleback, Gasterosteus aculeatus.</a> Copeia. 2001 (4), 936-944 (2001).</li><li>Berner, D., Moser, D., Roesti, M., Buescher, H., Salzburger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+architecture+of+skeletal+evolution+in+european+lake+and+stream+stickleback.">Genetic architecture of skeletal evolution in european lake and stream stickleback.</a> Evolution. 68 (6), 1792-1805 (2014).</li><li>Jamniczky, H. a., Barry, T. N., Rogers, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Eco-evo-devo+in+the+study+of+adaptive+divergence:+examples+from+threespine+stickleback+(Gasterosteus+aculeatus).">Eco-evo-devo in the study of adaptive divergence: examples from threespine stickleback (Gasterosteus aculeatus).</a> Integr. Comp. Biol. 55 (1), 166-178 (2015).</li><li>Magnuson, J., Heitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gill+raker+apparatus+and+food+selectivity+among+mackerels,+tunas,+and+dolphins.">Gill raker apparatus and food selectivity among mackerels, tunas, and dolphins.</a> Fish. Bull. 69 (2), 361-370 (1971).</li><li>Kahilainen, K. K., 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=The+role+of+gill+raker+number+variability+in+adaptive+radiation+of+coregonid+fish.">The role of gill raker number variability in adaptive radiation of coregonid fish.</a> Evol. Ecol. 25 (3), 573-588 (2011).</li><li>Arnegard, M. E., 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=Genetics+of+ecological+divergence+during+speciation.">Genetics of ecological divergence during speciation.</a> Nature. 511 (7509), 307-311 (2014).</li><li>Gross, H. P., Anderson, J. M., Gross, H. P., Anderson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geographic+variation+in+the+gillrakers+and+diet+of+European+threespine+sticklebacks,+Gasterosteus+aculeatus.">Geographic variation in the gillrakers and diet of European threespine sticklebacks, Gasterosteus aculeatus.</a> Copeia. 1984 (1), 87-97 (1984).</li><li>Hagen, D., Gilbertson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Geographic+variation+and+environmental+selection+in+Gasterosteus+aculeatus+L+in+the+Pacific+Northwest,+America.">Geographic variation and environmental selection in Gasterosteus aculeatus L in the Pacific Northwest, America.</a> Evolution. 26 (1), 32-51 (1972).</li><li>McPhail, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecology+and+evolution+of+sympatric+sticklebacks+(Gasterosteus):+morphological+and+genetic+evidence+for+a+species+pair+in+Enos+Lake,+British+Columbia.">Ecology and evolution of sympatric sticklebacks (Gasterosteus): morphological and genetic evidence for a species pair in Enos Lake, British Columbia.</a> Can. J. Zool. 62 (7), 1402-1408 (1984).</li><li>Schluter, D., McPhail, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecological+character+displacement+and+speciation+in+sticklebacks.">Ecological character displacement and speciation in sticklebacks.</a> Am. Nat. 140 (1), 85-108 (1992).</li><li>Robinson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trade+offs+in+Habitat-specific+foraging+efficiency+and+the+nascent+adaptive+divergence+of+sticklebacks+in+lakes.">Trade offs in Habitat-specific foraging efficiency and the nascent adaptive divergence of sticklebacks in lakes.</a> Behaviour. 137 (7), 865-888 (2000).</li><li>Glazer, A. M., Cleves, P. A., Erickson, P. A., Lam, A. Y., Miller, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+developmental+genetic+features+underlie+stickleback+gill+raker+evolution.">Parallel developmental genetic features underlie stickleback gill raker evolution.</a> Evodevo. 5 (1), (2014).</li><li>Miller, C. T., Glazer, A. M., 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=Modular+skeletal+evolution+in+sticklebacks+is+controlled+by+additive+and+clustered+quantitative+trait+loci.">Modular skeletal evolution in sticklebacks is controlled by additive and clustered quantitative trait loci.</a> Genetics. 197 (1), 405-420 (2014).</li><li>Glazer, A. M., Killingbeck, E. E., Mitros, T., Rokhsar, D. S., Miller, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+assembly+improvement+and+mapping+convergently+evolved+skeletal+traits+in+sticklebacks+with+Genotyping-by-Sequencing.">Genome assembly improvement and mapping convergently evolved skeletal traits in sticklebacks with Genotyping-by-Sequencing.</a> G3. 5, 1463-1472 (2015).</li><li>Wainwright, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+morphology+of+the+pharyngeal+jaw+apparatus.">Functional morphology of the pharyngeal jaw apparatus.</a> Fish Physiol. Fish Biomech. , 77-102 (2006).</li><li>Hulsey, C. D., Fraser, G. J., Streelman, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+development+of+complex+biomechanical+systems:+300+million+years+of+fish+jaws.">Evolution and development of complex biomechanical systems: 300 million years of fish jaws.</a> Zebrafish. 2 (4), 243-257 (2005).</li><li>Lauder, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+design+and+evolution+of+the+pharyngeal+jaw+apparatus+in+euteleostean+fishes.">Functional design and evolution of the pharyngeal jaw apparatus in euteleostean fishes.</a> Zool. J. Linn. Soc. 77, 1-38 (1983).</li><li>Fraser, G. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphology+and+kinetics+of+the+head+of+the+stickleback,+Gasterosteus+aculeatus.">Morphology and kinetics of the head of the stickleback, Gasterosteus aculeatus.</a> Trans. Zool. Soc. London. 32 (5), 311-416 (1974).</li><li>Meyer, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphometrics+and+allometry+in+the+trophically+polymorphic+cichlid+fish,+Cichlusomu+citrinelfum:+Alternative+adaptations+and+ontogenetic+changes+in+shape.">Morphometrics and allometry in the trophically polymorphic cichlid fish, Cichlusomu citrinelfum: Alternative adaptations and ontogenetic changes in shape.</a> J. Zool., Lond. 221, 237-260 (1990).</li><li>Huysseune, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenotypic+plasticity+in+the+lower+pharyngeal+jaw+dentition+of+Astatoreochromis+alluaudi+(Teleostei:+Cichlidae).">Phenotypic plasticity in the lower pharyngeal jaw dentition of Astatoreochromis alluaudi (Teleostei: Cichlidae).</a> Arch. Oral Biol. 40 (11), 1005-1014 (1995).</li><li>Muschick, M., Indermaur, A., Salzburger, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Convergent+Evolution+within+an+adaptive+radiation+of+cichlid+fishes.">Convergent Evolution within an adaptive radiation of cichlid fishes.</a> Curr. Biol. 22 (24), 2362-2368 (2012).</li><li>Cleves, 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=Evolved+tooth+gain+in+sticklebacks+is+associated+with+a+cis-regulatory+allele+of+Bmp6.">Evolved tooth gain in sticklebacks is associated with a cis-regulatory allele of Bmp6.</a> Proc. Natl. Acad. Sci. 111 (38), 13912-13917 (2014).</li><li>Ellis, N. 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=Distinct+developmental+and+genetic+mechanisms+underlie+convergently+evolved+tooth+gain+in+sticklebacks.">Distinct developmental and genetic mechanisms underlie convergently evolved tooth gain in sticklebacks.</a> Development. (142), 2442-2451 (2015).</li><li>Tucker, A. S., Fraser, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+developmental+diversity+of+tooth+regeneration.">Evolution and developmental diversity of tooth regeneration.</a> Semin. Cell Dev. Biol. 25-26, 71-80 (2014).</li><li>Erickson, P. A., Glazer, A. M., Cleves, P. A., Smith, A. S., Miller, C. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+developmentally+temporal+quantitative+trait+loci+underlie+convergent+evolution+of+increased+branchial+bone+length+in+sticklebacks.">Two developmentally temporal quantitative trait loci underlie convergent evolution of increased branchial bone length in sticklebacks.</a> Proc. R. Soc. 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. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+and+constraint+underlie+irreversibility+of+tooth+loss+in+cypriniform+fishes.">Selection and constraint underlie irreversibility of tooth loss in cypriniform fishes.</a> Proc. Natl. Acad. Sci. U. S. A. 111 (21), 7707-7712 (2014).</li><li>Pasco-Viel, E., 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=Evolutionary+trends+of+the+pharyngeal+dentition+in+Cypriniformes+(Actinopterygii+Ostariophysi).">Evolutionary trends of the pharyngeal dentition in Cypriniformes (Actinopterygii Ostariophysi).</a> PLoS One. 5 (6), e11293 (2010).</li></ol>

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.

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>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>
			<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 görselleştirme alternatif bir yöntem ile çift etiketleme sağlayan bir rodamin veya benzeri kırmızı filtre üzerinde fluoresces. floresan görüntüleme veya fenotiplendirme planlanan, floresan karanlıkta saklayın preps böylece, ışık hızlı bir şekilde kaybolur. Ventral görünümünden, solungaçları görüntülenmiştir ve pigmentasyon 43 ölçülebilir. Preps yıl% 100 gliserol saklanabilir. 4.jpg &quot;/&gt; Şekil 4:. Temsili dikenli brankiyal iskelet lekeli ve temizlenmiş brankiyal iskeletleri iki örnek gösterilmektedir. (A) aydınlık görüntü gösteren kemik kırmızı etiketli. (B) rodamin B filtre seti altında Floresan görüntü. diken, diş ve kemiklerin örnekleri sırasıyla düzeltme işaretleri, ok uçları ve yıldız işareti ile işaretlenmiştir. Ölçek çubukları = 2 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54056/54056fig4large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</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.

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

Nanyang Technological University

Research

JoVE Journal

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

Dissection of Drosophila Ovaries

Drosophila Yumurtalıkların diseksiyonu

Biyoloji

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.

Drosophila Larvaların NMJ Diseksiyon

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).

Brüt Diseksiyon Lobster Mide, Homarus 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.

Yetişkin Mouse Hipokampal dentat girus diseksiyonu

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.

Yetişkin Zebra balığı Organları diseksiyonu

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.

Diseksiyon ve boyanması Drosophila Larvaların Yumurtalıkların

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.

Yetişkin Zebra balığı Böbrek diseksiyonu

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.

Diseksiyon ve montajı<em&gt; Drosophila</em&gt; Pupa Göz Diskler

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.

Doğum sonrası Farelerde İşitsel Bulla diseksiyonu: Orta Kulak Kemikler ve histolojik analiz izolasyonu

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.

Diseksiyon ve DiceCT gizli ortaya başka veri evrim primat yüz ve ters

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