Agradecemos a Beninio Jomok por sua ajuda na criação de peixe-zebra. Este trabalho é financiado pelo NIH HL81753.

1. Preparação de slides e câmara umidificado <ol><li> A câmara úmida pode ser feita de uma caixa, como uma caixa de ponta esvaziado. Enrole tanto a câmara ea tampa com papel alumínio para proteger as amostras da luz. </li><li> Dentro da câmara, coloque uma pilha de toalhas de papel molhado para manter a umidade dentro da câmara, o que impedirá as amostras de coração de secagem. Definir uma microplaca pequena na parte superior da toalha de papel como um rack para os slides. </li><li> Desenhe linhas ou círculos sobre a superfície de uma lâmina de microscópio usando a caneta immEdge para fazer poços para amostras coração com uma dimensão de 5x5 milímetros. Deixe os slides seco. </li><li> Coloque as lâminas na câmara umidificada e adicionar 50uL formaldeído frescos 4% em cada poço. </li></ol> 2. Dissecção do coração embrionário <ol><li> Anestesiar o embrião de peixe em água contendo ovos E3 7 tricaina 0,4% para cerca de 1 minuto até que o peixe parar o movimento do corpo, mas ainda têm coração normalespancamento. </li><li> Coloque uma lâmina de microscópio côncava no palco de um microscópio de dissecção. Transferência dos embriões para o bem concaved. </li><li> Ajuste a combinação de luz do microscópio de dissecação claramente revelam o coração. Dependendo da preferência pessoal, pode ser tanto de campo escuro ou DIC-como condição de luz polarizada. </li><li> Limpe duas seringas de insulina com etanol 75%, depois seque. </li><li> Ajustar o microscópio de dissecação para maior ampliação e foco no coração. &#39;Fixar&#39; fisicamente um embrião através da inserção de uma ponta da agulha para a fronteira gema de somito perto da cabeça. Inserir outro perto da agulha para a região do coração e puxe o coração para fora rapidamente. Se o coração não é totalmente separado do embrião, cortar o tecido remanescente conectado entre o coração ea gema. </li><li> Ajustar o microscópio para menor ampliação e foco no coração separados. Use uma pipetteman 10μl e defini-lo ao volume 1μl. Aplicar a ponta do pipetteman a qualquer empate ou toque nacoração amostra, de modo que o coração isolado será dentro ou junto à superfície da ponta. </li><li> Mergulhe a ponta da pipeta na solução de formaldeído 4% em lâminas preparadas e comprimir o pistão da pipeta para libertar o coração para a solução. A tensão da superfície da água também ajuda a liberar o coração da ponta da pipeta para a solução. Coloque menos de 3 corações em cada poço. </li></ol> 3. Imunocoloração <ol><li> Fechar a câmara e colocá-lo em uma coqueteleira com um movimento de balanço suave. Fixar a amostra do coração por 20 minutos em temperatura ambiente. </li><li> Limpe a água na parte de trás das lâminas e colocar os slides no palco de um microscópio de dissecção para a troca de tampão. </li><li> Molhe a ponta de um pipetteman 10μl em uma região vazia em cada bem e manter a ponta na medida do possível a partir de amostras do coração. Tomar precauções para evitar o coração prender à ponta, já que os corações possam se mover com o fluxo de água quando a solução é arrastado para a ponta. Mudançaa localização da ponta, se necessário. </li><li> Descarte a solução em um tecido e evitar a produção de todas as bolhas na ponta, pois a amostra coração é facilmente perdido na presença de bolhas de ar. Manter as amostras coração parcialmente seco por um curto período de tempo pode ajudar a manter o coração ligado ao slides. </li><li> Adicionar 50μl PBST para cobrir a amostra coração e incubar na câmara umidificada em um movimento de balanço lento por 5 min. Repita o enxágüe mais uma vez. Após esta etapa, as amostras de coração pode ser armazenada na câmara úmida até 1 semana a 4 ° C. </li><li> Bloco da amostra coração com soro de ovelha 10% em PBST por 1 hora em temperatura ambiente. </li><li> Incubar a amostra coração com anticorpos primários, tais como a diluição de 1:200 β-catenina anticorpo e diluição de 1:200 mef2 anticorpo específico, em ovinos 10% de soro / PBST por 1 hora em temperatura ambiente. </li><li> Lavar a amostra coração com PBST três vezes (5 min. Cada) em temperatura ambiente. </li><li> Incubar a amostra com secundárioanticorpos, tais como a diluição de 1:1000 Alexa marcadas anti-rato e / ou anticorpos anti-coelho, em PBST para 0,5 horas. </li><li> Lavar as amostras coração com PBST três vezes (5 min. Cada) em temperatura ambiente. </li></ol> 4. Imagem <ol><li> Remover PBST e adicionar 10 mL de médio de montagem para cada poço. Rocha da câmara durante alguns minutos até que a solução se torna claro. </li><li> Remover a maior parte do meio de montagem e cobrir todos os poços em um slide com uma tampa de vidro de microscópio (24x50). </li><li> Coração imagem peixes usando Zeiss Axioplan 2 microscópio equipado com ApoTome (Carl Zeiss). </li></ol> 5. Resultados representante Um exemplo de uma imagem que revela membrana e núcleos de todos os cardiomiócitos zebrafish é mostrado na figura. 1. anticorpos mef2c e β-catenina foram usados ​​em conjunto para mancha amostras coração dissecados de 52 embriões zebrafish hpf. Enquanto mef2c manchas anticorpo os núcleos de cardiomiócitos, β-cateninaanticorpo revela a borda de cada célula 10/08. Ajustando o estágio do microscópio composto, as imagens das amostras do coração pode ser ajustado para a mesma orientação, o que permitirá a observação de curvatura externa e curvatura interna no coração 11. O número de cardiomiócitos total, o tamanho das células de cardiomiócitos e circularidade pode ser medido. Outro exemplo que revela a estrutura do sarcômero de um coração embrionário é mostrado na figura. 2. Realizamos imunocoloração usando F59, um anticorpo miosina, em um coração dissecado embrionárias. A rede miofibrila em um coração inteiro embrionárias podem ser claramente revelado no menor aumento, enquanto a banda estriada de filamento grosso pode ser revelado no maior ampliação (Figura 2) 6. <img alt="Figura 1" src="/files/ftp_upload/3510/3510fig1.jpg" /> Figura 1. Imunocoloração de mef2 (vermelho) e β-catenina (verde) para mostrar os núcleos e outlines de cardiomiócitos no ventrículo zebrafish. Barra de escala = 10μm <img alt="Figura 2" src="/files/ftp_upload/3510/3510fig2.jpg" /> Figura 2. Immnostaining de miosina para mostrar o filamento grosso em um ventrículo zebrafish em uma ou outra baixa ampliação (A) ou alta ampliação (B). Barra de escala = 10μm

<ol>
	<li>Shin, J. T., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+Zebrafish+to+human:+modular+medical+models.">From Zebrafish to human: modular medical models.</a> Annu. Rev. Genomics Hum. Genet. 3, 311-340 (2002).</li><li>Beis, D., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+biology:+following+the+zebrafish+trend.">In vivo cell biology: following the zebrafish trend.</a> Trends. Cell. Biol. 16, 105-112 (2006).</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>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nat. Genet. 26, 216-220 (2000).</li><li>Dahme, T., Katus, H. A., Rottbauer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fishing+for+the+genetic+basis+of+cardiovascular+disease.">Fishing for the genetic basis of cardiovascular disease.</a> Dis. Model Mech. 2, 18-22 (2009).</li><li>Huang, W., Zhang, R., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibrillogenesis+in+the+developing+zebrafish+heart:+A+functional+study+of+tnnt2.">Myofibrillogenesis in the developing zebrafish heart: A functional study of tnnt2.</a> Dev. Biol. 331, 237-249 (2009).</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=.">.</a> The Zebrafish Book. , (2007).</li><li>Buckingham, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle:+the+regulation+of+myogenesis.">Muscle: the regulation of myogenesis.</a> Curr. Opin. Genet. Dev. 4, 745-751 (1994).</li><li>Wheelock, M. J., Knudsen, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+and+associated+proteins.">Cadherins and associated proteins.</a> Vivo. 5, 505-513 (1991).</li><li>Sun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+hypertrophy+involves+both+myocyte+hypertrophy+and+hyperplasia+in+anemic+zebrafish.">Cardiac hypertrophy involves both myocyte hypertrophy and hyperplasia in anemic zebrafish.</a> PLoS One. 4, e6596-e6596 (2009).</li><li>Auman, 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=Functional+modulation+of+cardiac+form+through+regionally+confined+cell+shape+changes.">Functional modulation of cardiac form through regionally confined cell shape changes.</a> PLoS Biol. 5, e53-e53 (2007).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+zebrafish+essential+and+regulatory+myosin+light+chains+reduces+cardiac+function+through+distinct+mechanisms.">Depletion of zebrafish essential and regulatory myosin light chains reduces cardiac function through distinct mechanisms.</a> Cardiovasc. Res. 79, 97-108 (2008).</li></ol>

Não há conflitos de interesse declarados.

Em comparação com os métodos clássicos imunocoloração toda montagem, nosso método tem as seguintes vantagens. Em primeiro lugar, muito mais forte sinais fluorescentes podem ser obtidos de forma consistente, devido à penetração melhorada. Em todo o método de montagem de positividade, o tecido da pele densa que envolve o coração reduziu significativamente a penetração de muitos anticorpos, resultando em elevado ruído de fundo em todo o corpo. Este problema é especialmente grave para os embriões com mais ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td></tr><tr><td> tricaina </td><td> Orgânicos de pesquisa </td><td> 3007T </td></tr><tr><td> Formaldeído </td><td> Polysciences </td><td> 04018 </td></tr><tr><td> Seringa de insulina </td><td> Becton Dickinson </td><td> 329461 </td></tr><tr><td> Triton-X-100 </td><td> Sigma </td><td> T8532 </td></tr><tr><td> Anti-β-catenina anticorpo </td><td> Sigma </td><td> C7207 </td></tr><tr><td> Anticorpos anti-Mef2c </td><td> Santa Cruz Biotech </td><td> SC313 </td></tr><tr><td> F59 </td><td> Zfin </td><td></td></tr><tr><td> Anti-α-actinina anticorpos </td><td> Sigma </td><td> A7811 </td></tr><tr><td> Anti-anticorpo Ilk </td><td> Sinalização celular </td><td> # 3862 </td></tr><tr><td> Alexa flúor 568 cabra anti IgG de coelho </<html> td&gt; <td> Invitrogen </td><td> A11011 </td></tr><tr><td> Alexa flúor 488 cabra anti-rato IgG1 </td><td> Invitrogen </td><td> A21121 </td></tr><tr><td> Meio de montagem para fluorescência </td><td> Vetor </td><td> H-1200 </td></tr><tr><td> ImmEdge caneta </td><td> Vetor </td><td> H-4000 </td></tr><tr><td> Poli-L-lisina slides revestido </td><td> Microscopia eletrônica de ciências </td><td> 63410-01 </td></tr><tr><td> Tampa de vidro de microscópio </td><td> Pescador </td><td> 12-543-D </td></tr><tr><td> Concaved lâminas de microscópio </td><td> Pescador </td><td> 7-1305-8 </td></tr><tr><td> Microscópio de dissecção </td><td> Leica MZ95 </td><td></td></tr><tr><td> Microscópio composto </td><td> Zeiss </td><td> Axioplan2 </td></tr><tr><td> ApoTome </td><td> Zeiss </td><td></td></tr></tbody></table> PBST: 1 x PBS 0,5% Triton-X 100 ent &quot;&gt; tricaina 25X: 400 mg tricaina 97,9 mL DDH 2 O 2,1 mL (1M Tris PH9) Ajustar o pH para 7,0

immunostaining of dissected zebrafish embryonic heart

Embrião torna-se um popular paulistinha in vivo modelo de vertebrados para estudar o desenvolvimento de doenças cardíacas e coração humano, devido à sua embriologia e genética vantajoso 1,2. Cerca de 100-200 embriões estão prontamente disponíveis a cada semana a partir de um único par de peixes adultos. Os embriões que se desenvolvem transparente utero ex torná-los ideal para avaliar defeitos cardíacos 3. A expressão de qualquer gene pode ser manipulado através de morfolino tecnologia ou injeção de RNA 4. Além disso, a frente telas genética já gerou uma lista de mutantes que afetam diferentes perspectivas de cardiogenesis 5. Imunocoloração montar todo é uma técnica importante neste modelo animal para revelar o padrão de expressão da proteína alvo de um tecido especial 6. No entanto, imagens de alta resolução que pode revelar estruturas celulares ou subcelulares têm sido difíceis, principalmente devido à locat físicaion do coração e da pouca penetração dos anticorpos. Aqui, apresentamos um método para solucionar esses gargalos dissecando o coração primeiro e depois conduzir o processo de coloração na superfície de uma lâmina de microscópio. Para evitar a perda de amostras pequenas e coração para facilitar o manuseio solução, restringimos a amostra coração dentro de um círculo na superfície do microscópio slides puxado por uma caneta immEdge. Após a coloração, os sinais de fluorescência pode ser diretamente observado por um microscópio composto. Nosso novo método melhora significativamente a penetração de anticorpos, uma vez que um coração de um peixe embrionárias só consiste de camadas de células. Imagens de alta qualidade a partir de corações intactos podem ser obtidos dentro de um prazo muito reduzido para a procissão de embriões zebrafish com idades entre dia 2 a dia 6. Nosso método pode ser potencialmente estendido a outros órgãos dissecados mancha de qualquer peixe-zebra ou outros pequenos animais.

Watch this Scientific Journal Video about Immunostaining of Dissected Zebrafish Embryonic Heart at JoVE.com

Immunostaining of Dissected Zebrafish Embryonic Heart

Uma maneira rápida de realizar imunocoloração do coração embrionário zebrafish é descrito. Em comparação com toda a abordagem montagem imunocoloração, este método aumenta drasticamente a penetração dos anticorpos, o que permite obter imagens de alta resolução que revelam estruturas celulares / subcelulares no coração dentro de um tempo de processamento muito reduzido.

Imunomarcação de Dissecada Coração Zebrafish embrionárias

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

immunostaining-of-dissected-zebrafish-embryonic-heart

Research

JoVE Journal

Biology

17.6K visualizações.  Mayo Clinic College of Medicine. Uma maneira rápida de realizar imunocoloração do coração embrionário zebrafish é descrito. Em comparação com toda a abordagem montagem imunocoloração, este método aumenta drasticamente a penetração dos anticorpos, o que permite obter imagens de alta resolução que revelam estruturas celulares / subcelulares no coração dentro de um tempo de processamento muito reduzido.

Vídeo: Immunostaining of Dissected Zebrafish Embryonic Heart

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Vivem de imagem do cérebro Zebrafish embrionárias por microscopia confocal

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Biologia

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Tecido alvo quimeras embrionárias: Zebrafish celular Gástrula Transplante

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

Preparação de embriões de Microscopia Eletrônica do Drosophila Tubo cardíaco embrionário

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Microdissecção de tecidos oculares Zebrafish embrionárias

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Todo-mount Análise imuno-histoquímica para Vascularização embrionárias Limb pele: um sistema modelo para estudar Morfogênese Branching Vascular em Embrião

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

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

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

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

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

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 ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Um Sistema de<em&gt; Ex vivo</em&gt; A cultura de Pâncreas embrionárias

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-infligido Lesão de Zebrafish muscular esquelético embrionário

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

Análise de Desenvolvimento Embrionário e larval Zebrafish esqueléticos miofibras de Preparações dissociadas

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

A Novel<em&gt; Ex vivo</em&gt; Método de Cultura para o Rato coração embrionário

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...