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.

Todo o trabalho de peixe foi aprovado pelo Comitê Institucional de Animal Care and Use da Universidade da Califórnia-Berkeley (número de protocolo R330). A eutanásia foi realizada por meio de imersão em 0,025% tricaina-S tamponada com 0,1% de bicarbonato de sódio 39. Todos os passos são realizados à temperatura ambiente. 1. Preparação Nota: Execute as etapas 1,1-1,5 em tubos cônicos ou frascos de cintilação que pode selar firmemente e ser colocados na horizontal. Os peixes não precisam ser constantemente abalado, mas tente misturar a solução o mais rápido possível invertendo delicadamente ou sacudindo o rack de tubos ou frascos para expor todos os lados do peixe para a solução de coloração e permitir que mancha para penetrar no tecido uniformemente. Não coloque um grande lote de peixes em um agitador de plataforma, como o peso pesado do líquido vai quebrar o shaker. <ol><li> Fixar qualquer peixe recém-sacrificados ou peixe armazenado em etanol com 10% tamponada neutra de formalina (NBF) durante a noite. Como alternativa, use 4% paraformaldeído em solução 1x PBS em vez de NBF a 10%. Nota: Se extrair DNA, grampear uma pequena porção dos caudais ou barbatanas peitorais antes da fixação e armazenar em etanol. </li><li> Dispor de correção adequadamente em uma capa química e substituir com água da torneira (ou seja ~ pH 7,0) durante 2 horas. Evitar o uso de água deionizada, pois muitas vezes pode ser ácida e pode descalcificação óssea. </li><li> Retire a água e peixes mancha com 0,008% Alizarina S em 1% KOH em água por 24 horas. Para os peixes menos de 20 mm de comprimento padrão, utilize 0,004% Alizarina Red S. (Faça uma solução 100x (0,8%) de ações de Alizarina S, que pode então ser diluído). </li><li> Remova a mancha (colocar em um recipiente adequado de lixo na capa) e colocar peixes na água da torneira para alguns hr. Mudar a água como necessário até lavagem com água é mais clara. </li><li> Retire a água e coloque o peixe até 50% de glicerol, 0,25% KOH para limpeza suave e dissecção posterior. Nota: Este protocolo de coloração é modificado a partir de métodos anteriormente descritos 40,41. </li></ol><pclass = &quot;jove_title&quot;&gt; 2. dissecação Nota: Consulte a Figura 1 para uma revisão de cabeça relevante morfologia do esqueleto. <img alt="figura 1" src="/files/ftp_upload/54056/54056fig1.jpg" /> Figura 1:. Cabeça Stickleback morfologia do esqueleto Alizarina Red manchado cabeça Gasterosteus aculeatus fotografada com fluorescência sob um conjunto rodamina filtro B. morfologia útil é rotulado: Op = opérculo, Subop = subopercle, BSRs = branchiostegal raios, pré-operatório = preopérculo, Infraorb 1-3 = infraorbitários 1-3 (também chamados circumorbitals ou suborbitals), Dent = dentário, Premax = pré-maxila, Max = maxila , Nas = nasal, Lat. ethm = etmóide lateral, PSPH = paresfenóide, Fron = osso frontal. Para uma descrição mais detalhada da cabeça de esqueleto stickleback, consulte Anker (1974) 31. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54056/54056fig1large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior</a>esta figura. <ol><li> Deite o peixe plana (Figura 2A) e inserir uma pinça afiada # 5 de relojoeiro para o lado do olho com um ângulo de ~ 45 ° para perfurar a membrana que cobre o olho. </li><li> Descascar a membrana afastada, a partir do olho, semelhante à da casca de uma tampa de iogurte (Figura 2B). </li><li> Inserir uma pinça aberta atrás do olho, agarrar o nervo óptico por trás do olho, e remover o olho (Figura 2C). Não perfure o olho como ele vai vazar melanina. Se perfurado, a melanina pode ser lavado durante as etapas posteriores. </li><li> Repita do outro lado. </li><li> A partir do posterior, coloque uma lâmina de dissecação tesoura pequena sob a aba opérculo, lâmina de arrastar tesoura dorsal acima opérculo, em seguida, corte de tecidos moles até a órbita do olho (Figura 2D). Corte dorsal ao osso opérculo. </li><li> Cortar o osso frontal (dorsal para a cavidade ocular) (Figura 2E). </li><li> Cortar o osso paresfenóide linha média em torno de tEle centro das órbitas (Figura 2F). </li><li> corte opercle Repita no lado oposto. </li><li> Inserir uma pinça sob o opérculo e lentamente descascar o rosto para longe do corpo, aparando qualquer tecido mole ainda ligado (Figura 2G - H). Tome cuidado para não perturbar a primeira linha de rastros branquiais. <ol><li> Com uma pinça, retire as ceratohyals em ambos os lados do basihyal linha média, enquanto descascando e remover o esqueleto craniofacial anterior (toda a mandíbula, incluindo o dentário, pré-maxila, e da maxila; o esqueleto inteiro hióide incluindo o opérculo dérmica exterior, preopérculo, subopercle e raios branchiostegal e dorsal subjacente e elementos endocondrais ventral; e a parte anterior do crânio, incluindo o nasal, etmóide lateral e ossos infraorbital, ver figuras 1 e 2I). </li><li> espinhas pélvico pode ser dobrada para fora do corpo, e pode servir como um identificador para uma pinça para agarrar of quando presente. Espinhos encaixado. Para desbloquear, puxe coluna com uma pinça diretamente para fora do corpo do peixe, em seguida, dobre posteriormente para pressionar espinha plana contra peixes. </li></ol></li><li> Inserir fechada forceps posterior e ventral ao esqueleto branquial (logo abaixo tubo digestivo) e pinça de arrasto anteriormente, provocando além dos músculos restantes e ligamentos ligados ao esqueleto branquial (Figura 2J - K). </li><li> Usando as pontas de uma pinça fechadas, raspar os músculos que prendem a dorsal esqueleto branquial à caixa craniana ventral em um posterior à direção anterior (Figura 2 L). </li><li> Repita 2.9 e 2.10 do lado oposto. </li><li> Agarrar a base do tubo do intestino e puxar anteriormente para remover o esqueleto e intestino tubo branquial (Figura 2 M - N). </li><li> Separa-se o tubo do intestino posterior, fazendo um corte perpendicular ao final do quinto ceratobranquial (Figura 2O </strong&gt;). </li><li> Depois de remover quaisquer fragmentos de osso restantes da caixa craniana do lado dorsal do esqueleto branquial, inserir uma tesoura para o cesto branquial para fazer um corte dorsal (corte anterior para posterior) entre os conjuntos bilaterais de placas dorsal do dente (Figura 3A - D). Certifique-se de corte é centrada para evitar danificar as placas dorsal dente. </li><li> Fazer dois cortes laterais rasa no lúmen do intestino de borracha na extremidade posterior do esqueleto branquial (extremidade anterior do tubo do intestino) para ajudar na abertura do esqueleto branquial (Figura 3E). </li><li> Coloque todos os peixes e pedaços de tecido para um frasco e colocar o esqueleto branquial para um tubo de microcentrífuga com 50% de glicerol, 0,25% de KOH para continuar a limpeza suave, ou 100% de glicerol, se não mais de compensação é necessário. frascos de etiquetas e tubos com um identificador único para que eles possam ser rastreados. A quantidade de purificação necessário é em grande parte uma função do tamanho do peixe, peixe adulto grande (mais de 40 mm de comprimento padrão) normalmente exigem compensação adicional. </li></ol><img alt="Figura 2" src="/files/ftp_upload/54056/54056fig2.jpg" /> Figura 2:. Stickleback branquial esqueleto dissecção Alizarina Red manchado peixes Gasterosteus aculeatus pronto para dissecção. O olho é despigmentada da extensa clareira. As setas azuis indicam a direcção do movimento. (A) Vista lateral da cabeça stickleback, anterior está à direita. (B) A remoção de membrana que cobre o olho. (C) A remoção do olho. (D) Dorsal corte acima do opérculo. Corte ósseo (E) frontal. (F) parasphenoide corte. (G - I) A remoção do esqueleto facial. (J) A remoção do esqueleto branquial conexões de tecidos moles ventral. (K - L) Remoção de dorsais conexões esqueleto branquial. (&lt;strong&gt; M - N) A remoção do esqueleto branquial. (O) Separa-se o tubo digestivo do esqueleto branquial. Veja os passos 2.1 a 2.16 para mais detalhes. Barra de escala = 5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54056/54056fig2large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a> 3. branquial esqueleto Re-coloração (se necessário) <ol><li> Para mancha mais escura do esqueleto branquial ou tecido mais clara, remover glicerol a 50%, solução de KOH 0,25% e lava-se com KOH a 1%, duas vezes (uma lavagem de cinco minutos, seguida por uma segunda lavagem 24 h, agitando horizontalmente num agitador de plataforma). </li><li> Retirar 1% de KOH e re-mancha com 0.008% vermelho de alizarina S em 1% de KOH durante 24 h. </li><li> Remover manchas e substituir com 1% de KOH durante 24 h. </li><li> Remoção da solução de KOH e substituir com 50% de glicerol, 0,25% KOH. </li></ol> 4. Montagem branquial Skeleton <ol><li> Remover esqueleto branquiala partir de 50% de glicerol, 0,25% de KOH ou 100% de glicerol e local perto da parte inferior de um x 60 milímetros lamela de vidro com 22 milímetros lado dorsal voltada para cima (Figura 3F). Adicionam-se algumas gotas de 50% de glicerol, 0,25% de KOH ou 100% de glicerol em cima de esqueleto branquial. Se a transição a partir de 50% de glicerol, 0,25% de KOH e 100% de glicerol, solução mudança em um tubo de microcentrífuga e agitar durante&gt; 5 min antes da montagem para equilibrar tecido. </li><li> Rolar para fora duas pequenas bolas de massa de modelar e colocar em cada extremidade da lâmina de cobertura para agir como espaçadores. </li><li> Vagamente colocar uma segunda lamela por cima com pressão suficiente para achatar o esqueleto branquial anterior (Figura 3G). </li><li> Peel abrir a aba dorsal esquerda, incluindo placas de dentes dorsais, alise, e deslize entre as lamelas (Figura 3h). </li><li> Técnica Repita com a aba dorsal direita e empurrar toda esqueleto branquial longe da borda da lamela (Figura 3I). <ol><li> Alterarnativamente, segure ambas as abas abertas dorsais com uma pinça e coloque cuidadosamente a lamela na, achatando o esqueleto branquial em um movimento suave. </li><li> Alternativamente, montar o esqueleto branquial de cabeça para baixo em uma lamela, splaying cada lado dorsal lateralmente assim que a gravidade não permite que o esqueleto branquial para fechar volta. Em seguida, cubra com o segundo x 60 milímetros lamela de vidro 22 mm e inverter prep. Nota: Diferentes técnicas de montagem tendem a trabalhar melhor ou pior para cada indivíduo. Tente cada um e ver o que se sente mais confortável. </li></ol></li><li> Pressione levemente na lamela superior para achatar bolas de argila suficiente para manter o esqueleto branquial montado plana, mas tome cuidado para não esmagar o espécime. <ol><li> Durante o processo de montagem, ceratobranchials pode girar e obscurecer uma fileira de rastros. Remediar esta situação através de pinça entre as lamelas e re-orientar o ceratobranchials ou toda esqueleto branquial deslizante. </li></ol></li><li> Loja prepara apartamento em bandejas de slides no rotemperatura om. Montada em 100% de glicerol, preps pode ser armazenada entre lamelas em ponte por pelo menos uma década. forceps limpas e tesouras com isopropanol ou etanol e dicas de cobertura. </li></ol><img alt="Figura 3" src="/files/ftp_upload/54056/54056fig3.jpg" /> Figura 3:. Plano de montagem do esqueleto branquial Manipulação e montagem do esqueleto branquial é mostrado. As setas azuis indicam a direcção do movimento. Lado para cima (A) branquial esqueleto dorsal. (B - D) Rotação e incisão entre as placas dorsal dente. (E) corte lateral em tecido mole para abrir ainda mais a base do tubo digestivo. Esqueleto (F) Branquial colocado na parte inferior de uma lamela pronto para montagem. (L) Em segundo lugar lamela colocada na metade anterior do esqueleto branquial (acima placas dentárias dorsais). (H - I)montagem plana do esqueleto branquial abrindo abas placa dorsal dente e deslizando entre duas lamelas. Veja os passos 4.1 até 4.6 para obter mais detalhes. Barra de escala = 5 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54056/54056fig3large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</a>

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J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ancient+gene+network+is+co-opted+for+teeth+on+old+and+new+jaws.">An ancient gene network is co-opted for teeth on old and new jaws.</a> PLoS Biol. 7 (2), e1000031 (2009).</li><li>Stock, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+dentition+in+comparative+context.">Zebrafish dentition in comparative context.</a> J. Exp. Zool. B. Mol. Dev. Evol. 308, 523-549 (2007).</li><li>Liem, K., Greenwood, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+functional+approach+to+the+phylogeny+of+the+pharyngognath+teleosts.">A functional approach to the phylogeny of the pharyngognath teleosts.</a> Am. Zool. 21 (1), 83-101 (1981).</li><li>Anker, G. 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.

Uma incrível quantidade de diversidade existe na cabeça do esqueleto entre os vertebrados, especialmente entre os peixes. Em muitos casos, essa diversidade facilita diferentes estratégias de alimentação 1-4, e pode envolver grandes alterações tanto padronização craniofacial externo e interno. O esqueleto branquial está localizado internamente na garganta de um peixe e rodeia a maior parte da cavidade bucal. O esqueleto branquial é composta por 5 segmentos série homóloga, o anterior quatro dos quais suportam as guelras. Juntos, esses cinco segmentos funcionar como uma interface entre o peixe e sua comida 5. Variação em uma infinidade de traços incluindo rakers Gill, dentes da faringe e ossos branquial contribuir para forrageamento eficiente em diferentes tipos de alimentos. Sticklebacks tenham sido submetidos a uma radiação adaptativa após formas oceânicas ancestrais colonizaram lagos de água doce e riachos em todo o hemisfério norte. A mudança na dietade pequeno zooplâncton no oceano para presas maiores em água doce resultou em variação trófica dramática em várias características craniofaciais 6. Enquanto muitos estudos têm-se centrado nas diferenças craniofaciais externas em sticklebacks 7 - 13, importantes alterações craniofaciais evoluir repetidamente no esqueleto branquial interno. A capacidade de criar híbridos férteis entre as populações stickleback morfologicamente distintas proporciona uma excelente oportunidade para mapear a base genética das alterações evoluíram para o esqueleto branquial. Um traço trófica de importância ecológica é a padronização dos rastros branquiais, ossos dérmicos periódicas que revestem as faces anterior e posterior dos ossos branquial e são usados ​​para filtrar presas. Os peixes que normalmente se alimentam de pequenos presas tendem a ter mais tempo e mais densamente Gill espaçadas rakers em comparação com os peixes que se alimentam de presas maiores 14,15. Variação de rastros branquiais tem sido relatada tanto within e entre espécies 14-19, e os aspectos de emalhar raker padronização contribuir para nichos tróficos e de fitness 16. Décadas de pesquisa têm amplamente documentado número branquiais e variação de comprimento em sticklebacks threespine 17 - 21; no entanto, esses estudos geralmente se concentram na primeira linha de rastros branquiais. Um trabalho recente tem mostrado modularidade no controle genético do número branquiais em todo o esqueleto branquial 22,23 e em uma única linha no raker Gill espaçamento 23 e comprimento de 24 destacando a importância de estudar mais de remar um ou uma única raker Gill para entender o base genética do desenvolvimento da redução branquiais. Uma segunda característica trófica de ambos importância ecológica e biomédica é a padronização dos dentes da faringe. Os dentes em peixes pode ser localizada tanto na mandíbula oral e no esqueleto branquial, conhecido como os dentes da faringe. dentes orais são usados ​​principalmente para prey capturar enquanto os dentes da faringe são utilizados para a mastigação e presa manipulação 25-27. Ambos os conjuntos formam através de mecanismos de desenvolvimento compartilhados e são considerados developmentally homólogo 28. Modularidade interessante ocorre pela qual algumas espécies, como o peixe-zebra, a falta dentes da faringe oral e dorsal 29, enquanto outras espécies têm múltiplas ceratobranchials dentadas, pharyngobranchials, e às vezes dentada basihyal e hypobranchials 30. Em sticklebacks, dentes da faringe são encontrados ventrally no quinto ceratobranquial e dorsal sobre a anterior e posterior pharyngobranchials 31. Cinemática sobre alimentação stickleback mostrar a mandíbula oral é utilizado principalmente para a captura de presas e facilitar a alimentação de sucção 9 deixando a mastigação para a mandíbula da faringe. Em ciclídeos, inferior a morfologia da faringe mandíbula varia dramaticamente 32,33 e foi mostrada para ser adaptável e correlacionada com nicho trófico 34. multipopulações de água doce stickleback PLE têm evoluído aumentos dramáticos na faringe ventral número de dentes 23,35,36. Um trabalho recente demonstrou que a base genética do desenvolvimento deste ganho dente evoluído é em grande parte distinta em duas populações derivadas independentemente de sticklebacks de água doce 36. Ao contrário de dentes de mamíferos, peixes regenerar os dentes de forma contínua ao longo da vida adulta 37. Ambas as populações de água doce dentadas elevados previamente descritos têm evoluído uma taxa acelerada substituição de dentes, fornecendo um sistema de vertebrado rara para estudar a base genética de regeneração 36. Uma terceira característica trófica que evoluiu repetidamente no sticklebacks de água doce é mais ossos epibranchial e ceratobranquial, os branquial homólogos arco segmentar da mandíbula superior e inferior, respectivamente 38. Mais longos ossos branquial conferem um cavidade bucal maior e provavelmente são adaptáveis ​​para permitir presas maiores para ser consumed. Além disso, em outros peixes, ossos epibranchial são importantes para a depressão das placas de dentes dorsal da faringe 25. Como rastros branquiais e dentes da faringe, os ossos branquiais são internas e, portanto, difícil de visualizar ou quantificar facilmente. Aqui apresentamos um protocolo detalhado para dissecar e de montagem fixa o esqueleto branquial, permitindo fácil visualização e quantificação de uma variedade de importantes características craniofaciais. Embora este protocolo descreve uma dissecção stickleback, este mesmo método funciona com uma variedade de outros peixes.

<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> Este protocolo resulta em um esqueleto montado branquial dissecados e plana (Figura 4), ​​onde uma variedade de importantes características tróficas podem ser quantificados. Do ponto de vista dorsal, todas as linhas de rastros branquiais, todas as placas da faringe dente, e quase todos os ossos branquiais podem ser facilmente visualizados e quantificados 22 - 24,35,36,38,42. Vermelho de Alizarina S reage também em uma ou rodamina filtro vermelho semelhante permitindo que a dupla marcação com outros marcadores (por exemplo, GFP transgénico 42) e um método alternativo de visualização. Fluorescência desaparece rapidamente na luz, para armazenar preps no escuro se imagens fluorescentes ou fenotipagem é planejado. A partir de uma vista ventral, as brânquias pode ser visualizada e a coloração quantificada 43. Preps podem ser armazenados em 100% de glicerol durante anos. 4.jpg &quot;/&gt; Figura 4:. Stickleback Representante esqueleto branquial Dois exemplos de esqueletos branquial manchadas e apuradas são mostrados. (A) Brightfield imagem que mostra óssea marcado vermelho. (B) imagem fluorescente sob um conjunto rodamina filtro B. Exemplos de rastros, dentes e ossos são rotulados com carets, pontas de seta e asteriscos, respectivamente. Barras de escala = 2 mm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54056/54056fig4large.jpg" target="_blank">Por favor clique aqui para ver uma versão maior desta figura.</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.

Dissecção e pay-per-montagem do Threespine Stickleback branquial 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 ...

dissection-flat-mounting-threespine-stickleback-branchial

Nanyang Technological University

Research

JoVE Journal

Biology

9.7K visualizações.  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. 

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

Dissection of Drosophila Ovaries

Dissecção da Drosophila Ovários

Biologia

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 larval Dissection MNJ

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).

Dissecação bruta do Estômago da lagosta, 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.

Dissecção do giro denteado do hipocampo de rato adulto

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.

Dissecção de Órgãos do Zebrafish Adulto

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.

Dissecção e coloração de Drosophila Larval Ovários

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.

Dissecção do rim Zebrafish Adulto

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.

Dissecção e montagem de<em&gt; Drosophila</em&gt; pupais Discos Eye

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.

Dissecção da Auditivo Bulla em ratos pós-natais: Isolamento dos ossos do ouvido médio e da análise histológica

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.

Reverter a dissecção e dados DiceCT revelar outra forma escondida na evolução do primata rosto

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