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>

<ol>
	<li>Hsu, H. J., Lin, G., Chung, B. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+early+development+of+zebrafish+interrenal+glands+and+pronephros:+differential+control+by+wt1+and+ff1b.">Parallel early development of zebrafish interrenal glands and pronephros: differential control by wt1 and ff1b.</a> Development. 130, 2107-2116 (2003).</li><li>To, T. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pituitary-interrenal+interaction+in+zebrafish+interrenal+organ+development.">Pituitary-interrenal interaction in zebrafish interrenal organ development.</a> Mol Endocrinol. 21, 472-485 (2007).</li><li>Liu, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interrenal+organogenesis+in+the+zebrafish+model.">Interrenal organogenesis in the zebrafish model.</a> Organogenesis. 3, 44-48 (2007).</li><li>Cravioto, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+inherited+variant+of+the+3+beta-hydroxysteroid+dehydrogenase-isomerase+deficiency+syndrome:+evidence+for+the+existence+of+two+isoenzymes.">A new inherited variant of the 3 beta-hydroxysteroid dehydrogenase-isomerase deficiency syndrome: evidence for the existence of two isoenzymes.</a> J Clin Endocrinol Metab. 63, 360-367 (1986).</li><li>Lachance, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+human+3+beta-hydroxysteroid+dehydrogenase/delta+5-delta+4-isomerase+gene+and+its+expression+in+mammalian+cells.">Characterization of human 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase gene and its expression in mammalian cells.</a> J Biol Chem. 267, 3551 (1992).</li><li>Simard, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+biology+of+the+3beta-hydroxysteroid+dehydrogenase/delta5-delta4+isomerase+gene+family.">Molecular biology of the 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase gene family.</a> Endocr Rev. 26, 525-582 (2005).</li><li>Lin, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+zebrafish+hsd3b+genes+are+distinct+in+function,+expression,+and+evolution.">Two zebrafish hsd3b genes are distinct in function, expression, and evolution.</a> Endocrinology. 156, 2854-2862 (2015).</li><li>Grassi Milano, E., Basari, F., Chimenti, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adrenocortical+and+adrenomedullary+homologs+in+eight+species+of+adult+and+developing+teleosts:+morphology,+histology,+and+immunohistochemistry.">Adrenocortical and adrenomedullary homologs in eight species of adult and developing teleosts: morphology, histology, and immunohistochemistry.</a> Gen Comp Endocrinol. 108, 483-496 (1997).</li><li>Chai, C., Liu, Y. W., Chan, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ff1b+is+required+for+the+development+of+steroidogenic+component+of+the+zebrafish+interrenal+organ.">Ff1b is required for the development of steroidogenic component of the zebrafish interrenal organ.</a> Dev Biol. 260, 226-244 (2003).</li><li>Liu, Y. W., Gao, W., Teh, H. L., Tan, J. H., Chan, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prox1+is+a+novel+coregulator+of+Ff1b+and+is+involved+in+the+embryonic+development+of+the+zebra+fish+interrenal+primordium.">Prox1 is a novel coregulator of Ff1b and is involved in the embryonic development of the zebra fish interrenal primordium.</a> Mol Cell Biol. 23, 7243-7255 (2003).</li><li>Chai, C., Liu, Y. W., Chan, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ff1b+is+required+for+the+development+of+steroidogenic+component+of+the+zebrafish+interrenal+organ.">Ff1b is required for the development of steroidogenic component of the zebrafish interrenal organ.</a> Dev. Biol. 260, 226-244 (2003).</li><li>Chou, C. W., Zhuo, Y. L., Jiang, Z. Y., Liu, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hemodynamically-regulated+vascular+microenvironment+promotes+migration+of+the+steroidogenic+tissue+during+its+interaction+with+chromaffin+cells+in+the+zebrafish+embryo.">The hemodynamically-regulated vascular microenvironment promotes migration of the steroidogenic tissue during its interaction with chromaffin cells in the zebrafish embryo.</a> PLoS One. 9, e107997 (2014).</li><li>Liu, Y. W., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelium+is+required+for+the+promotion+of+interrenal+morphogenetic+movement+during+early+zebrafish+development.">Endothelium is required for the promotion of interrenal morphogenetic movement during early zebrafish development.</a> Dev Biol. 297, 44-58 (2006).</li><li>Jin, S. W., Beis, D., Mitchell, T., Chen, J. N., 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=Cellular+and+molecular+analyses+of+vascular+tube+and+lumen+formation+in+zebrafish.">Cellular and molecular analyses of vascular tube and lumen formation in zebrafish.</a> Development. 132, 5199-5209 (2005).</li><li>Proulx, K., Lu, A., Sumanas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+vasculature+in+zebrafish+forms+by+angioblast+cluster-derived+angiogenesis.">Cranial vasculature in zebrafish forms by angioblast cluster-derived angiogenesis.</a> Dev Biol. 348, 34-46 (2010).</li><li>Perner, B., Englert, C., Bollig, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wilms+tumor+genes+wt1a+and+wt1b+control+different+steps+during+formation+of+the+zebrafish+pronephros.">The Wilms tumor genes wt1a and wt1b control different steps during formation of the zebrafish pronephros.</a> Dev Biol. 309, 87-96 (2007).</li><li>Chou, C. W., Chiu, C. H., Liu, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibronectin+mediates+correct+positioning+of+the+interrenal+organ+in+zebrafish.">Fibronectin mediates correct positioning of the interrenal organ in zebrafish.</a> Dev Dyn. 242, 432-443 (2013).</li><li>Chiu, C. H., Chou, C. W., Takada, S., Liu, Y. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+fibronectin+signaling+requirements+of+the+zebrafish+interrenal+vessel.">Development and fibronectin signaling requirements of the zebrafish interrenal vessel.</a> PLoS One. 7, e43040 (2012).</li></ol>

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 border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Confocal microscope</td>
			<td style="width:187px;">Carl Zeiss&#160;</td>
			<td style="width:72px;">LSM510&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma</td>
			<td>D8418&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>USB</td>
			<td>US16374</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Fetal Calf Serum&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30073</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-Nicotinamide adenine dinucleotide hydrate</td>
			<td>Sigma</td>
			<td>N1636</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-Nitro blue tetrazolium</td>
			<td>Promega</td>
			<td>S380C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nusieve GTG</td>
			<td>Lonza</td>
			<td>50081</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylthiourea</td>
			<td>Sigma&#160;</td>
			<td>P7629</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Sigma</td>
			<td>P4417-100Tab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PYREX Spot Plate</td>
			<td>Corning</td>
			<td>7220-85</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reef Salt</td>
			<td>AZOO</td>
			<td>AZ28001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trans-Dehydroandrosterone</td>
			<td>Sigma</td>
			<td>D4000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td>Sigma</td>
			<td>P9416</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td>Leica</td>
			<td>VT1000M</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

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Captura de Reparo de Tecidos em Zebrafish larvas com a Time-lapse Brightfield lupa estereoscópica

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Biologia do Desenvolvimento

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Análise de Zebrafish larvas Músculo Esquelético Integrity com Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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 ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

Protocolos para a visualização de esteroidogênicas Órgãos e seus órgãos interativa com imunocoloração na mosca da fruta<em&gt; Drosophila melanogaster</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Uma nova técnica de transplante de almofada de gordura mamária para visualizar a geração de vasos de células vasculares endoteliais vasculares

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Imagem ao vivo do mouse Fusão do palato secundário

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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 ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Celular agregação ensaios para avaliar a vinculação do entalhe drosófila com Trans-ligantes e sua inibição pelo Cis-ligantes

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

Seguindo os movimentos de tecido endocárdico através de célula Photoconversion no embrião Zebrafish

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualizando o nó e placa Notochordal em Gastrulating embriões de rato usando microscopia eletrônica e imunofluorescência de montagem inteira

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...