Graças à Marie Birne, Tsiola Areti, Estevez Jaymie e Uribe Arelys. Este trabalho foi financiado em parte pela CUNY Research Foundation e Instituto Médico Howard Hughes Grant Programa de Verão para Estudantes de Graduação (SPUR) e RO3 NIH 41702-00-01. A pesquisa foi apoiada também por Queens College e alguns dos experimentos foram feitos em equipamentos a partir do Núcleo Facility for Imaging, Biologia Celular e Molecular no Queens College.

Peixe-zebra foram levantadas utilizando protocolos padrão 13 no Biotério Queens College a 28 ° C. Pares de peixes individuais ou acasalamentos grupo deve ser pré-formados e os embriões resultantes limpos e armazenados em um 28 ° C incubadora. Saúde dos peixes deve ser examinado diariamente. Em cinco dias pós-fertilização (DPF), o peixe deve ser separado em tanques a uma densidade de peixes 10-15 e entraram na creche facilidade de peixe. Para o protocolo aqui apresentados foram coletados em zebrafish larva por estágios adultos: 15, 30, 45, 60, 90, 180 dpf. 1. Peixe-zebra coleção assunto e fixação <ol><li> Retire o peixe do tanque de habitat usando net e coloque em 0,2% tricaina para anestesiar. </li><li> Coloque os peixes anestesiados em água gelada por 15 minutos para euthanize. </li><li> Faça paraformaldeído a 4% fresco (PFA) em 1x tampão fosfato (PBS). </li><li> Incubar peixes coletados em PFA 4% por 1-2 horas à temperatura ambiente, em seguida, durante a noite a 4 ° C numa placa de Petri envolto em parafilme. Isso mantém o plano de peixe e depois faz a dissecação muito mais fácil. </li><li> Após a fixação, lavar duas vezes em PBS com Tween (PBT). Peixe pode ser manter em PBT, a 4 ° C por até 10 dias. </li></ol> 2. Medição de peixes e dissecção <ol><li> Peixes único lugar em meia petri prato cheio de PBS fresco e colocá-lo em seu lado direito. </li><li> Medida de comprimento de peixes do focinho à base da cauda (o pedúnculo caudal), não incluindo aletas de cauda, ​​utilizando um paquímetro digital de milímetro (Figura 1). Este é o comprimento padrão (SL). </li><li> Cada peixe pode ser atribuído um número / combinação de letras para permitir que um indivíduo eo coração, resultando dissecados para ser rastreados para análise posterior. Dados, incluindo o número de ações dos pais, a idade de fixação, medições SL, foram inseridos em uma planilha e organizado com base nesses rótulos. </li><li> Antes da dissecação, etiqueta tiras tubo de PCR com o número de rastreamento atribuído a preencher com PBT ou outra solução apropriada para análise posterior. </li><li> Orient lado ventral do peixe em placa de petri, enquanto a estabilização do corpo com pinças segurando a cabeça entre os olhos e as guelras (Figura 2). </li></ol> Peixes menores (menores que 12 milímetros SL): <ol start="4"><li> a) Utilizando uma pinça afiada, ou uma micro-agulha em um suporte de pino, retire os músculos peitorais e as aletas do corpo para revelar o coração. O movimento constante do fórceps durante a dissecção pode causar correntes no PBT que movem o peixe. Isto é particularmente problemático para peixes menores, portanto, um micro-agulha pode ser usado para remover músculos peitorais e barbatanas e abrir a cavidade do corpo. </li><li> a) delicadamente usar a pinça para colher o coração para fora da cavidade debaixo do átrio. Alternadamente, em alguns peixes que você pode ser capaz de remover o coração puxando para fora bulboso eo resto do coração se seguirão. Tenha cuidado com a localização do átrio, se usar esta técnica, pois ele pode ser facilmente danificada. Se houver tecido extra que vem com o coração, que é fino e pode ser apagada depois. </li><li> a) Se o peixe contém um marcador fluorescente cardíacos, tais como Tg (myl7: GFP) twu34, use âmbito fluorescentes para dissecção e verificar a remoção do coração pelo florescimento (Figura 3). </li><li> a) Uma vez que o coração é removido da cavidade, use a pinça para segurar o coração e uma micro-agulha para remover o tecido não-cardíacas extra e do revestimento do epicárdio de fora do coração. </li></ol> Peixes maiores (mais de SL 12mm): <ol start="4"><li> b) Usando Primavera tratado micro-tesoura, faça três incisões de menos de 1mm de profundidade: 1 - corte transversal através do 2 brânquias - corte transversal no ventre anterior 3 - corte sagital no lado ventral conectando dois cortes (Figura 4). </li><li> b) Utilizando uma pinça afiada remove os músculos peitorais e as aletas do corpo para abrir a cavidade do corpo e revelar o tecido prateado do pericárdio (Figura 5A). Remover este tecido de pericárdio eo coração se tornará visível (Figura 5B). </li><li> b) Use a tesoura para cortar micro-artéria ligado ao arteriosus bulbosa, situada superior ao coração. Uma vez que esta artéria é cortada, coloque as pontas fórceps sob o átrio, use a pinça para colher o coração para fora da cavidade. Se houver tecido extra que vem com o coração, que é fino e pode ser removido mais tarde. </li><li> b) Uma vez que o coração é removido da cavidade, use os dois pinça para segurar o coração e remover o tecido extra, ou um par de pinças para segurar o coração e uma micro-agulha para remover extra, o tecido não-cardíacas. O forro do epicárdio, evidente pela sua pigmentação preta, pode ser retirado a partir do exterior do coração, se desejar delicadamente raspando o tecido. </li></ol> 3. Fotografia de corações <ol><li> Prepare uma 23g / L de ágar nutriente placa de petri e cubra ágar solidificado com PBS. </li><li> Usando a pinça, faça um buraco no agar que permite que o coração para descansar na orientação desejada para fotografar. </li><li> Remove o coração do tubo de PCR, segurando a ponta do bulbo com a pinça com firmeza. Coloque o coração no prato agar. </li><li> Orientar o coração em agar para fotografar. A amostra pode ser rodado depois de cada imagem para todas as orientações possíveis (Figura 6). </li><li> Use o tempo de exposição de luz sobrecarga e curto para fotografar o coração. Fazer ajustes para a qualidade da luz usando a exposição necessário para obter a melhor fotografia. </li></ol> 4. Resultados representativos: Um exemplo de um coração bem dissecados limpa é mostrado em orientações múltiplas na Figura 6. Cada coração vai ter algumas diferenças na morfologia geral, mas o coração deve ser completa, contendo um ventrículo intacto, átrio e arteriosus bulbosa. Alguns exemplos desta variação na morfologia geral são mostrados na Figura 7. O preto manchado tecido epicárdico também pode removido do coração. <img src="/files/ftp_upload/3165/3165fig1.jpg" alt="Figura 1"/> Figura 1. Meça os peixes do focinho à pedúnculo caudal usando uma câmera digital milímetros paquímetro. <img src="/files/ftp_upload/3165/3165fig2.jpg" alt="Figura 2"/> Figura 2. Segure o peixe com uma pinça entre os olhos e guelras. <img src="/files/ftp_upload/3165/3165fig3.jpg" alt="Figura 3"/> A Figura 3 cardíaca marcador fluorescente específica transgênicos, como Tg (myl7: GFP). Twu34, mostrado aqui, pode auxiliar na visualização corações dissecados de peixes menores. <img src="/files/ftp_upload/3165/3165fig4.jpg" alt="Figura 4"/> Figura 4 Três incisões para remover os músculos peitorais e da pele: 1 - corte transversal através das brânquias 2 - corte transversal no ventre anterior 3 - corte sagital no lado ventral conectando ambas as cortes.. <img src="/files/ftp_upload/3165/3165fig5.jpg" alt="Figura 5"/> . Figura 5 letras Black Label as câmaras do coração: arteriosus bulbosa (ba), ventrículo (v), e no átrio (a). A-A cavidade superior do corpo está aberto para mostrar o tecido prateado do pericárdio. B-Após a remoção do pericárdio, o coração é visível e facilmente removidos. <img src="/files/ftp_upload/3165/3165fig6.jpg" alt="Figura 6"/> Figura 6. Orientações diferentes do coração são mostrados. Letras pretas rótulo as câmaras do coração: arteriosus bulbosa (ba), ventrículo (v), e no átrio (a). Setas indicam Cinzento localização e orientação do coração, em referência à sua localização normal no corpo: anterior (A), posterior (P), dorsal (D), ventral (V), esquerda (L) e direito (R). Barra de escala indica 400 micras. A-ventral vista do coração B-dorsal vista do coração C-lateral vista do lado esquerdo D-lateral vista do lado direito <img src="/files/ftp_upload/3165/3165fig7.jpg" alt="Figura 7"/> Figura 7. Morfologia de corações de tamanhos diferentes é mostrada. Letras pretas rótulo as câmaras do coração: arteriosus bulbosa (ba), ventrículo (v), e no átrio (a). Barra de escala indicada 400 m. AC-Hearts peixes formulário com menos de 12 milímetros SL DF-Hearts peixes formulário com SL maior que 12 mm

<ol>
	<li>Ahmad, N., Long, S., Rebagliati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+southpaw+joins+the+roster:+the+role+of+the+zebrafish+nodal-related+gene+southpaw+in+cardiac+LR+asymmetry.">A southpaw joins the roster: the role of the zebrafish nodal-related gene southpaw in cardiac LR asymmetry.</a> Trends Cardiovasc Med. 14, 43-49 (2004).</li><li>Armstrong, E. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+endothelial+cell+signaling+and+differentiation.">Heart valve development: endothelial cell signaling and differentiation.</a> Circ Res. 95, 459-470 (2004).</li><li>Bakkers, J., Verhoeven, M. C., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+zebrafish+heart:+from+left-right+axis+specification+to+epithelial+tissue+morphogenesis.">Shaping the zebrafish heart: from left-right axis specification to epithelial tissue morphogenesis.</a> Dev Biol. 330, 213-220 (2009).</li><li>Glickman, N. S., Yelon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+development+in+zebrafish:+coordination+of+form+and+function.">Cardiac development in zebrafish: coordination of form and function.</a> Semin Cell Dev Biol. 13, 507-513 (2002).</li><li>Heicklen-Klein, A., McReynolds, L. J., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+zebrafish+model+to+study+GATA+transcription+factors.">Using the zebrafish model to study GATA transcription factors.</a> Semin Cell Dev Biol. 16, 95-106 (2005).</li><li>Schoenebeck, J. J., Yelon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+cardiac+development:+Advances+in+imaging+add+new+dimensions+to+the+utility+of+zebrafish+genetics.">Illuminating cardiac development: Advances in imaging add new dimensions to the utility of zebrafish genetics.</a> Semin Cell Dev Biol. 18, 27-35 (2007).</li><li>Beis, 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+cellular+analyses+of+zebrafish+atrioventricular+cushion+and+valve+development.">Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development.</a> Development. 132, 4193-4204 (2005).</li><li>Lepilina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+epicardial+injury+response+supports+progenitor+cell+activity+during+zebrafish+heart+regeneration.">A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration.</a> Cell. 127, 607-619 (2006).</li><li>Wills, A. A., Holdway, J. E., Major, R. J., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+addition+of+new+myocardial+and+epicardial+cells+fosters+homeostatic+cardiac+growth+and+maintenance+in+adult+zebrafish.">Regulated addition of new myocardial and epicardial cells fosters homeostatic cardiac growth and maintenance in adult zebrafish.</a> Development. 135, 183-192 (2008).</li><li>Parichy, D. M., Elizondo, M. R., Mills, M. G., Gordon, T. N., Engeszer, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+table+of+postembryonic+zebrafish+development:+staging+by+externally+visible+anatomy+of+the+living+fish.">Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish.</a> Dev Dyn. 238, 2975-3015 (2009).</li><li>Huang, C. J., Tu, C. T., Hsiao, C. D., Hsieh, F. J., Tsai, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+a+myocardium-specific+GFP+transgene+reveals+critical+regulatory+elements+in+the+cardiac+myosin+light+chain+2+promoter+of+zebrafish.">Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.</a> Dev Dyn. 228, 30-40 (2003).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Book,+A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">The Zebrafish Book, A guide for the laboratory use of zebrafish (Danio rerio).</a> , (1995).</li></ol>

Não há conflitos de interesse declarados.

Estes métodos para dissecção do coração do peixe zebra pós-embrionário de peixes permite a remoção completa de um coração danificado. Embora essa técnica é simples e fornece guias para cortes externos que irão evitar danos ao coração, uma mão firme é essencial para peixes menores. Um passo fundamental durante a dissecção é identificar o pericárdio prateado como o coração está diretamente dentro desse saco. Visualizando o pericárdio fornece um importante marcador par...

<table border="1"><tbody><tr><th> Material </th><th> Companhia </th><th> Número de catálogo </th><th> Comentários (opcional) </th></tr><tr><td> Paraformaldeído </td><td> EMD </td><td> PX0055 </td><td> .04g/mL Em PBS </td></tr><tr><td> Fórceps # 55 </td><td> FST </td><td> 11295-51 </td><td></td></tr><tr><td> Micro-tesoura </td><td> FST </td><td> 15005-08 </td><td></td></tr><tr><td> Titular Pin </td><td> FST </td><td> 26016-12 </td><td></td></tr><tr><td> Micro-agulhas </td><td> FST </td><td> 10130-10 </td><td></td></tr><tr><td> Agar nutriente </td><td> BBL </td><td> 11472 </td><td> 23g / L de H 2 O destilada </td></tr><tr><td> Placas de Petri </td><td> BD Falcon </td><td> 351029 </td><td></td></tr><tr><td> PCR 8 tubo de tiras </td><td> EUA Scientific </td><td> 1402-2500 </td><td></td></tr><tr><td> Caliper Digital </td><td> Fisher Scientific </td><td> 14-648-17 </td><td></td></tr></tbody></table>

heart dissection in larval, juvenile and adult zebrafish, danio rerio

Peixe-zebra tornou-se um organismo modelo benéfico e prático para o estudo do desenvolvimento embrionário do coração (ver comentários recentes 1-6), no entanto, o trabalho de examinar pós-embrionário, através do desenvolvimento adulto cardíaca tem sido limitada 7-10. Examinar a morfologia mudança do coração amadurecimento e envelhecimento são restritas pela ausência de técnicas disponíveis para o estadiamento e isolar os corações juvenis e adultos. A fim de analisar o desenvolvimento do coração mais tempo de vida do peixe, dissecamos corações zebrafish em estágios numerosos e fotografá-los para posterior análise 11. As características morfológicas do coração podem ser facilmente quantificadas e corações individuais podem ser posteriormente analisado por uma série de métodos standard. Zebrafish crescer a taxas variáveis ​​e maturação se correlaciona melhor com o tamanho do peixe do que a idade, assim, pós-fixação, nós comprimento dos peixes fotografia e medida como um indicador de maturação dos peixes. Este protocolo explica duas distintas, tamanho técnicas de dissecção dependente de peixe-zebra, que vão desde larvas de comprimento padrão de 3,5 mm (SL) com corações de comprimento ventrículo 100μm (VL), para adultos, com SL de 30mm e 1mm VL ou maior. Larvas de peixes e adultos têm corpo bastante distintos e morfologia do órgão. Larvas não são apenas significativamente menor, eles têm menos pigmentos e cada órgão é visualmente muito difíceis de identificar. Por este motivo, usamos técnicas de dissecção distintas. Usamos pré-dissecção procedimentos de fixação, como descobrimos que os corações dissecados imediatamente após a eutanásia tem uma morfologia mais variável, com balão muito solto e, como átrios em comparação com os corações retirados fixação seguintes. O peixe fixadas antes dissecção, manter vivo na morfologia e posição de câmara (dados não mostrados). Além disso, para fins de demonstração, aproveitamos do coração (miocárdio) específicos GFP transgênicos Tg (myl7: GFP) twu34 (12), o que nos permite visualizar todo o coração e é particularmente útil nos primeiros estágios de desenvolvimento quando o cardíaco morfologia é menos distinta dos tecidos adjacentes. Dissecção do coração faz uma análise mais aprofundada da biologia celular e molecular subjacentes ao desenvolvimento e maturação do coração utilizando hibridização in situ, imuno-histoquímica, extração de RNA ou outros métodos de análise mais fácil no pós-embrionário zebrafish. Este protocolo irá proporcionar uma técnica valiosa para o estudo da maturação de desenvolvimento cardíacas e envelhecimento.

Watch this Scientific Journal Video about Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio at JoVE.com

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Um método, claro padronizados para dissecção e isolamento do coração zebrafish em vários estágios de desenvolvimento são descritos. Técnicas de anotação e quantificação também são discutidos.

Dissecção do coração em larval, Zebrafish Juvenil e Adulto, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

heart-dissection-in-larval-juvenile-and-adult-zebrafish-danio-rerio

Research

JoVE Journal

Biology

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

21.4K visualizações.  Queens College, City University of New York. Um método, claro padronizados para dissecção e isolamento do coração zebrafish em vários estágios de desenvolvimento são descritos. Técnicas de anotação e quantificação também são discutidos.

Vídeo: Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissecção do CNS Larval em Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

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 and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

Dissecação bruta e Multa de Ouvido Interno Epitélio sensorial em adultos Zebrafish ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

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 ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

Em Eletroporação vivo de Morpholinos na Retina Zebrafish Adulto

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

Cuidados e manutenção regular de um peixe-zebra (<em&gt; Danio rerio</em&gt;) Laboratório: Uma Introdução

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

In Vivo Modelagem do Morbid Genoma Humano utilizando Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavaging Zebrafish Adulto

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

Dissecção do coração em larval, Zebrafish Juvenil e Adulto, Danio rerio

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