Agradecemos Prof. Christoph Englert e Prof. Didier Stainier para presentear a Tg (wt1b: GFP) LI1 e Tg (kdrl: EGFP) s843 estirpes, respectivamente, e do Mecanismo de Taiwan Zebrafish núcleo para fornecer Tg (kdrl: mCherry) CI5. Este estudo foi apoiado por subsídios do Ministério da Ciência e Tecnologia (96-2628-B-029-002-MY3, 101-2313-B-029-001, 102-2628-B-029-002-MY3 de Taiwan, 102- 2321-B-400-018).

Declaração de Ética: Todos os procedimentos experimentais no peixe-zebra foram aprovados pelo Comitê Cuidado e Uso Institucional animal da Universidade Tunghai (Aprovação IRB NO 101-12.) E realizado em conformidade com as orientações aprovadas. 1. As soluções de reserva para 3β-HSD Actividade Enzimática A coloração <ol><li> Prepare trans-dehydroandrosterone [10 mg / ml em sulfóxido de dimetilo (DMSO)]. </li><li> Prepare β-nicotinamida adenina dinucleótido hidrato (1,2 mg / ml em tampão fosfato 0,1 M, pH 7,2). </li><li> Prepare a nicotinamida (vitamina B3, 50 mg / ml em H2O). </li><li> Prepare de tetrazólio azul nitro-4 (50 mg / ml em 70% de dimetilformamida). </li><li> Alíquotas todos esses componentes em tubos de microcentrífuga e armazenar a -20 ° C. NOTA: Em nossa experiência, ciclos repetidos de congelamento e descongelamento das alíquotas de todas as soluções acima mencionadas não afectam a intensidade da coloração de atividade 3β-HSD. </li></ol> 2. Whole-montagem 3β-HSD Atividade Coloração NOTA: A actividade enzimática 3β-HSD é detectável nos embriões de peixe-zebra tão cedo quanto 28 horas após a fertilização (HPF) quando cultivadas a 28,5 ° C, o que é consistente com o aparecimento da expressão de ARNm de hsd3b1 2,7. O protocolo de toda a montagem de coloração de actividade 3β-HSD neste estudo baseia-se num método anteriormente descrito 8 e determinou-se ser bem sucedido no peixe-zebra desenvolver até 7 dias após a fertilização 9. Para analisar o microambiente vascular do tecido esteroidogênica interrenal, conjunto de montagem 3β-HSD de coloração de actividade deve ser aplicado às linhas transgénicas expressando fluorescência específica do endotélio, tal como TG (kdrl: EGFP) s843 14 e Tg (kdrl: mCherry) CI5 15 . Para analisar o tecido esteroidogênica interrenal considerando o desenvolvimento do rim zebrafish, atividade 3β-HSD coloração pode ser apliEd à Tg (wt1b: GFP) LI1 peixe 16, onde a formação do glomérulo e pronephric túbulos é delineado. <ol><li> Tratar o peixe-zebra desenvolvimento, incubando-as em 0,03% em água feniltioureia ovo (sal de 0,06 mg / L de recife em água desionizada), a partir de uma fase de entre 12 e 24 hpf, para inibir a formação de pigmentos. </li><li> Fix embriões dechorionated ou larvas em (a) 4% de paraformaldeído (PFA) em solução salina tamponada com fosfato (PBS) suplementado com 0,1% de Tween 20 (PFAT) durante 1 h à temperatura ambiente (TA) ou (B) 2% PFAT durante a noite a 4 ° C ou durante 2 horas a 4 ° C, seguido por 4 horas à temperatura ambiente. Cuidado: PFA é tóxico por inalação e em contacto com a pele. É destrutivo para a pele, membranas mucosas, olhos, e tracto respiratório superior. </li><li> Lava-se a embriões 4x durante 10 min com PBS suplementado com 0,1% de Tween 20 (PBST) a TA. NOTA: É crítico para não secar as amostras ao alterar as soluções de lavagem. </li><li> Recém preparar o staining solução antes das reacções (Tabela 1). Prepare, vortex, centrifugação e partes A e B, em tubos separados. NOTA: A adição de todos os componentes em linha reta em um único tubo iria causar precipitação grave. </li><li> Adicionar toda a solução da Parte A para toda a solução da Parte B, misturar bem para preparar a solução de coloração, e distribuir imediatamente 1 ml em cada tubo de microcentrífuga contendo embriões fixados e lavadas. </li><li> Enrole tubos de microcentrífuga com folha de alumínio para proteger da luz e o local de cada um rotor ou de uma plataforma de agitação para agitação suave à temperatura ambiente. </li><li> Empiricamente determinar o tempo de reação, monitorando sinais cromogénicos por meio de microscopia. precipitações leves irão desenvolver ao longo do tempo; No entanto, eles não irão interferir com o processo de reacção. NOTA: Para os embriões fixada em 28 hpf, a coloração, a 25 ° C durante 4 h gera um sinal claro no tecido interrenal. </li><li> Parar a reacção através de lavagem 4 x durante 10 minutos em PBST. EuF mais nenhuma análise imuno-histoquimica (IHC) é necessário, pós-corrigir os embriões corados com 4% PFAT durante 60 min à TA. Caso contrário, armazenar os embriões foram lavados a 4 ° C até uso posterior. </li><li> Para a microscopia todo-mount, limpar os embriões corados, pós-fixados, e lavado com 50% de glicerol em PBS, orientá-los com o lado dorsal para cima em um slide, e observar com um microscópio vertical sob iluminação de campo claro. NOTA: Para fazer uma imagem de alta resolução da morfologia do tecido interrenal, a análise do embrião 3β-HSD atividade-coradas e deyolked plana de montagem é recomendada. Como o tecido esteroidogênica interrenal está posicionado logo acima do saco vitelino, uma análise ventral plano de montagem do tecido interrenal permite uma observação direta e clara da morfologia interrenal 17. </li></ol> 3. IHC Análise de 3β-HSD Embriões manchado de Atividade NOTA: Para analisar o microambiente vascular do steroitecido dogenic, os embriões manchado de atividade 3β-HSD com um repórter fluorescente específica do endotélio transgênicos são submetidos a deslocamentos vibratome seccionamento e subsequente análise IHC 12,18. <ol><li> Pós-fixação <ol><li> Pós-corrigir os embriões 3β-HSD actividade coradas e lavou-se com 2% de PFA suplementado com 1% de Triton X-100 durante 1 hora a 4 ° C e lavar 4x durante 10 min em PBS contendo 1% de Triton X-100 (PBSTx) à TA. </li></ol></li><li> Incorporação <ol><li> Prepare a 4% de agarose de baixo ponto de fusão dissolvendo o de agarose em PBS a 95 ° C, em tubos de microcentrífuga alíquota, e armazenar a 4-8 ° C. </li><li> Derreter-se uma alíquota de 4% de agarose de baixo ponto de fusão a 70 ° C durante 10 min, e em seguida manter a aliquota a 47 ° C. Incorporar cada embrião no fundido de 4% de agarose de baixo ponto de fusão em um tampão individual de um tubo de microcentrífuga de 1,5 ml, que serve como um molde, e deixa-se arrefecer até solidificar. </li></ol></li><li> vibratome Seccionamento <ol><li> Cole um pedaço de fita de papel para a p secolatform de vibratome. </li><li> Remover o bloco de agarose endurecida do molde por uma lâmina de barbear. Aplicar supercola para a fita de papel na plataforma, e fixar o bloco de agarose na fita de papel, com o lado anterior da amostra virada para cima. Aparar o bloco de agarose a uma forma de prisma trapezoidal, com aproximadamente 4 mm e de 8 mm em cada lado das facetas superiores e inferiores, respectivamente. </li><li> Lavar o bloco de agarose contendo amostra com PBS frio suplementado com 0,5% de Triton X-100 (0,5% PBSTx), utilizando um conta-gotas. </li><li> Corte o bloco de agarose em 100 mm seções de acordo com o manual de instruções do vibratome. Constantemente lavar o bloco de agarose para evitar a secagem. À medida que cada fatia é cortada, removê-lo a partir da vibratome usando uma agulha G 26 da seringa, e transferir para uma placa de ponto frio contendo 0,5% PBSTx (500 ul / poço). </li><li> Recolher as secções de tecido de atividade positivos 3p-HSD, verificando sob um microscópio de dissecação, e transferir as seções por um piponta pette com a sua extremidade de corte (cerca de 1 mm de diâmetro interior na abertura) numa placa de 96 poços contendo 0,5% frio PBSTx (150 ul / poço). As amostras de tecido geralmente dissociar-se do bloco de agarose após este passo. </li></ol></li><li> Permeabilização e Lavagem <ol><li> Lavam-se as fatias 6x durante 15 min em 0,5% PBSTx à TA, 10 min em PBS, e 4x durante 10 minutos em PBS suplementado com 1% de albumina de soro de bovino / 1% de DMSO / 0,1% de Triton X-100 (PBDTx). </li></ol></li><li> Bloqueio <ol><li> Incubam-se as fatias para o soro 1 h em 2% de vitelo fetal (FCS) em PBDTx à TA. </li></ol></li><li> Anticorpo primário <ol><li> Incubam-se as fatias durante 1,5 dias em PBDTx contendo um anticorpo primário (por exemplo, policlonal de coelho anti-fibronectina humana e de ratinho anti-humano quinase de adesão focal), a 4 ° C. </li></ol></li><li> Lavando <ol><li> Lavam-se as fatias 6x durante 10 min em PBDTx à TA. </li></ol></li><li> Bloqueio <ol><li> Incubam-se as fatias durante 1 h em 2% de FCS em PBDTx à TA. </li></ol></li><li> Anticorpo secundário <ol><li> Incubam-se as fatias para 1,5 dias em PBDTx contendo um anticorpo secundário (anticorpo de cabra anti-coelho conjugada com fluoróforo ou anti-IgG de ratinho) a 4 ° C. </li></ol></li><li> Lavando <ol><li> Lavam-se as fatias 6x durante 10 min em PBDTx à TA e 4x durante 10 min em PBST à TA. NOTA: Para a análise microscópica confocal, limpar as amostras com 50% de glicerol em PBS. </li></ol></li></ol> 4. Análise confocal <ol><li> Observe todo o embrião montagem ea seção por um microscópio confocal equipado com 10X / 0.5 e 20X / 0,75 lente objetiva. </li><li> Para a detecção simultânea da fluorescência e da coloração 3β-HSD, definir o modo de multicanal como se segue: 488 nm do laser de árgon e 505-530 nm, filtro de banda para a detecção de GFP; 543 nm do laser de Hélio-Néon e 560-615 nm, filtro de banda para a detecção de mCherry; e transmitida canal de luz para a detecção de coloração 3β-HSD.O tamanho pinhole é uma unidade Airey, ea resolução é de 1024 x 1024 pixel. </li><li> Montar as pilhas Z (com 6 intervalo mm) como uma projeção em 3D, e fundir a projeção e o campo brilhante, usando o built-in software confocal. </li></ol>

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The authors have no competing financial interests to declare.

A intensidade do sinal da actividade 3β-HSD aumentado ao longo do curso de reacção. Sinais claros de actividade 3β-HSD foram detectados após 4 horas de reacção para as fases de 28 HPF em diante. No entanto, a duração da reacção requer a determinação empírica, dependendo do objetivo do ensaio. Nos casos em que a coloração requer processamento durante a noite, um fundo ligeiramente azulado tende a desenvolver sobre as amostras. Este problema pode ser ultrapassado pelo aumento da duração de fixação ...

A glândula adrenal, um componente crucial do eixo hipotálamo-hipófise-adrenal, segrega esteróides e coordena a homeostase esteróides e na resposta do organismo ao estresse. A glândula adrenal compreende o córtex exterior, que segrega esteróides de um modo específico da zona, e medula interna, que sintetiza catecolaminas. A glândula interrenal em teleósteos é o homólogo da glândula supra-renal em mamíferos e é composto de células cromafins e interrenais esteroidogênicas, que são equivalentes funcionais do córtex adrenal e medula, respectivamente 1-3. Estudos realizados utilizando o modelo de peixe-zebra têm relatado que ambas as linhagens celulares esteroidogênicas e cromafins são formados por mecanismos moleculares e celulares altamente semelhantes aos mamíferos em 1,2. Por isso, o peixe-zebra é um modelo potencialmente poderosa para o estudo de doenças genéticas, controlo neuroendócrino, e sistemas de biologia do eixo hipotálamo-hipófise-adrenal (interrenal). <p class = &quot;jove_content&quot;&gt; na glândula adrenal, 3β-HSD catalisa a conversão da progesterona a partir de pregnenolona, 17α-hidroxiprogesterona de 17α-hydroxypregnelolone, e androstenodiona a partir de desidroepiandrosterona 4,5. 3β-HSD é essencial para biosynthesizing todas as classes de esteróides hormonais, nomeadamente progesterona, glucocorticóides, mineralocorticóides, androgénios, estrogénios e. Os dois humana 3β-HSD isozimas HSD3B1 e HSD3B2 são diferencialmente expressos 6. HSD3B1 é expressa na placenta e nos tecidos periféricos, enquanto que HSD3B2 é expresso no córtex adrenal e das gónadas. HSD3B1 humano e HSD3B2 são co-ortólogos de hsd3b1 peixe-zebra, que é expressa no tecido interrenal e gónadas adultos; HSD3B2 peixe-zebra é um gene maternalmente expressa cuja transcrições desaparecer antes organogênese 7. O protocolo do ensaio de actividade de toda a montagem 3β-HSD enzimática para peixe-zebra foi desenvolvido através da modificação do método Levy,s descritas por Milano et al. , Em cortes congelados de oito espécies de teleósteos 8. Devido à permeabilidade dos tecidos e a transparência óptica do peixe-zebra desenvolvimento, todo-montagem 3β-HSD histoquímica pode ser usada com sucesso para o embrião do peixe-zebra fixa e larvas e especificamente delinear os tecidos interrenais diferenciadas. Este ensaio sensível e rápida foi aplicado a vários mutantes e morphants demonstrando diferentes tipos de dysmorphogenesis interrenal. A actividade 3β-HSD interrenal está ausente no embrião quando as especificações do tecido interrenal é interrompida através de um knockdown específica do factor de transcrição Ff1b e diminui à medida que a diferenciação interrenal é afectada por um knockdown da coregulator Ff1b Prox1 9,10. Notavelmente, a actividade 3β-HSD pode ser detectada em mutantes com defeitos graves da fase inicial, tais como uma cabeça de alfinete e de olhos estrabismo, onde o 3β-HSD histoquímica delineia como a migração celular é afectada interrenal 11. A diferenciação do tecido interrenal não é comprometida, mesmo na ausência total de sangue e vasculatura. Portanto, como os sinais derivados do endotélio moldar o órgão interrenal em desenvolvimento pode ser determinado 12,13. Em geral, este ensaio histoquímico foi usado com êxito para estudar especificação, diferenciação e migração de células esteroidogénicas no modelo de peixe-zebra. Portanto, ele deve ser um eficiente e uma ferramenta confiável para as telas genéticos ou químicos visando as disfunções dos órgãos supra-renais e interrenais.

<table><tbody><tr><td>Confocal microscope</td><td>Carl Zeiss&amp;nbsp;</td><td>LSM510&amp;nbsp;</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>USB</td><td>US16374</td><td></td></tr><tr><td>Hyclone Fetal Calf Serum&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>SH30073</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide hydrate</td><td>Sigma</td><td>N1636</td><td></td></tr><tr><td>4-Nitro blue tetrazolium</td><td>Promega</td><td>S380C</td><td></td></tr><tr><td>Nusieve GTG</td><td>Lonza</td><td>50081</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Phenylthiourea</td><td>Sigma&amp;nbsp;</td><td>P7629</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>P4417-100Tab</td><td></td></tr><tr><td>PYREX Spot Plate</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>Reef Salt</td><td>AZOO</td><td>AZ28001</td><td></td></tr><tr><td>trans-Dehydroandrosterone</td><td>Sigma</td><td>D4000</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000M</td><td></td></tr></tbody></table>

visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

Para determinar como as esteroidogênicas codevelops tecido interrenais com o glomérulo renal pronephric e sua vasculatura nascente, o ensaio de actividade enzimática 3β-HSD foi realizada na dupla transgénico Tg (wt1b: GFP) li1; Tg (kdrl: mCherry) CI5 embrião em 34 HPF (Figura 1). Nesta fase, o tecido esteroidogênica actividade positiva 3β-HSD está localizado à direita da linha média e caudal imediatamente para o glomérulo renal ...

Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

A glândula interrenal no peixe-zebra é a contrapartida teleósteos da glândula supra-renal de mamífero. Este protocolo apresenta a forma de realizar a desidrogenase de 3-hidroxiesteróides β (Δ 5-4 isomerase; 3β-HSD) ensaio da actividade enzimática, que detecta as células diferenciadas esteroidogênicas no peixe-zebra desenvolvimento.

Visualizando o Tissue interrenais Steroidogenic e sua Vascular Microenvironment em Zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

visualizing-interrenal-steroidogenic-tissue-its-vascular

Research

JoVE Journal

Developmental Biology

6.9K visualizações.  Tunghai University.  A glândula interrenal no peixe-zebra é a contrapartida teleósteos da glândula supra-renal de mamífero. Este protocolo apresenta a forma de realizar a desidrogenase de 3-hidroxiesteróides β (Δ 5-4 isomerase; 3β-HSD) ensaio da actividade enzimática, que detecta as células diferenciadas esteroidogênicas no peixe-zebra desenvolvimento. 

Vídeo: Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

Análise de Desenvolvimento de rim Zebrafish com imagem Time-lapse Usando um microscópio de dissecação Equipado para Seccionamento Optical

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Biologia do Desenvolvimento

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

Imunocoloração fosfo-epitopos em ciliadas Órgãos da Whole Mount embriões Zebrafish

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Química

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

Dissecção de larval Zebrafish gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

In vivo Imaging e Quantitation da resposta angiogênica hospedeira em xenoenxertos de tumor de zebrafish

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Pesquisa do câncer

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Visualização de células Multiciliated no Zebrafish através de um protocolo combinado de toda montagem fluorescente In Situ da hibridação e imunofluorescência

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Modelos de lesão adulto Zebrafish para estudar os efeitos de prednisolona na regeneração de tecido ósseo

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

Quantificando o tamanho do fígado em zebrafish larval usando microscopia de campo brilhante

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

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 ...

Biologia

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Estudando a Diabetes através dos olhos de um peixe: Microdissection, visualização e análise do tg(fli:EGFP) adulto vasculatura da retina de Zebrafish

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Medicina

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Monte Preparação Plano de observação e análise de amostras de embriões Zebrafish manchado por inteiro Monte<em&gt; Em situ</em&gt; Hibridização

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