We would like to thank Dr. Robert Angerer for his careful reading and editing of the manuscript. NIH R15HD088272-01 as well as the Office of Research and Development, and Department of Biological Sciences at Mississippi State University provided support for this project to RCR.

1. Rendimento Morpholino Estratégia de alto design <ol><li> Identificar um gene (s) de interesse (por exemplo, abordagem do gene candidato, análise de cis-regulatórias, RNA-Seq e ou telas diferenciais / proteômica). </li><li> Use genômica, transcriptomic, e dados de expressão de genes disponíveis em sites atualizados com freqüência (por exemplo SpBase http://www.echinobase.org 20 e S. purpuratus Genome Pesquisa http: ///urchin.nidcr.nih.gov/blast/index .html) para determinar se o perfil de expressão espaço-temporal sobrepõe-se com o mecanismo de desenvolvimento em questão. Se não houver dados disponíveis expressão é, em seguida, gerar iniciadores qPCR e / ou um anti-sentido na sonda in situ para avaliar o padrão de expressão do gene. </li><li> Depois de determinar o padrão de expressão espacial e temporal, obter a sequência de DNA a partir dos sites genômicos. <ol><li> Para a geração de oligonucleótidos morfolino-tradução de bloqueio, obter sequência óf a &quot;região não-traduzida (5 &#39;5 UTR) directamente a montante do codão de iniciação de expresso Sequence Tag (EST) bases de dados disponíveis no SpBase ou S. purpuratus Genoma sites de busca. Se esta informação não está disponível para o gene de interesse, em seguida, executar 5 &#39;RACE. </li><li> Para projetar um morfolino-blocking emenda, procure a seqüência genômica incluindo os exons e introns usando a ferramenta de andaime BLASTN em SpBase ou S. purpuratus Genome ferramenta de busca. </li></ol></li><li> Use o site oligonucleotídeo concepção http://oligodesign.gene-tools.com a fim de projetar a sequência par morfolino 25 base desejada. <ol><li> Para um bloqueio de morfolino-translacional, utilizar a sequência de ARNm a partir de 5 &#39;para 3&#39; como a fonte da sequência alvo de translação. Incluir sequência a 5 &#39;UTR (cerca de 70 nucleótidos a montante do sítio do início da transcrição), mais 25 bases da região de codificação (a jusante do local de início) com o codão de iniciação Wi acentuadath parênteses (ATG). </li><li> Para um bloqueio de morfolino-splicing, seleccionar intrão-exão ou uma sequência de fronteira exão-intrão e incluem 50 bases (25 bases da sequência de exão e 25 bases da sequência do intrão) em torno das regiões de fronteira. Digitalizar o genoma com a sequência morfolino para verificar que a sequência é única. </li></ol></li></ol> 2. A microinjecção de morfolino oligonucleótidos <ol><li> Prepare 3 soluções de mM dos oligonucleï¿½idos morfolino pela adição de 100 mL de água livre de nuclease para dentro do frasco morfolino 300 nmol. NOTA: Não use DEPC água tratada para a re-suspensão porque dietilpirocarbonato pode danificar o morfolino. </li><li> Para a primeira rotação reconstituição sobre o frasco contendo a solução de oligonucleótido durante 30 s à velocidade máxima (14000 - 16000 xg), vortex brevemente, aquece-se a 65 ° C durante 5 a 10 min, vortex brevemente, e manter o estoque morfolino no quarto temperatura durante pelo menos 1 h. solução de morfolino<html> s são armazenados de -20 ° C a 4 ° C. </li><li> Preparar a solução de injecção contendo oligonucleótidos morfolino na concentração desejada. Esta solução normalmente contém 20% de glicerol ou KCl 125 mM como um transportador e 15% de FITC-Dextrano (isotiocianato de f luoresceina de dextrano 10.000 MW de 2,5 mg Solução de estoque / mL). outros conjugados de dextrano fluorescente FITC-Dextrano e são rotineiramente usadas para identificar embriões injectados por microscopia de epifluorescência. soluções para injecção armazenar a -20 ° C. </li><li> Aquece-se a solução de morfolino a 65 ° C num bloco de aquecimento ou banho de água, durante pelo menos 2-5 minutos. </li><li> Resumidamente girar a solução de morfolino por 30 s em velocidade máxima (14000 - 16000 xg), vortex durante 1 min e centrifuga-se a toda a velocidade (14.000 - 16.000 xg) durante pelo menos 10 min. </li><li> Coloque as agulhas de injecção com a solução morfolino. Para um protocolo de microinjeção detalhadas, por favor consulte Stepicheva e Song, 2014 21 e Felicidades e Ettensohn de 2004&quot;&gt; 22. </li></ol> 3. Fixação e in situ Protocolo às 24 horas pós-fertilização (hpf) em S. purpuratus Embriões NOTA: Este protocolo é modificado a partir Arenas-Mena et al. , 2000 23 e Sethi et ai. De 2014 24. <ol><li> Fixação <ol><li> Adicione algumas gotas de fixador (veja abaixo) para poços contendo embriões, misture delicadamente por pipetagem, e permitir-lhes para resolver. Remover a solução fixadora, e, em seguida, misturar embriões com duas lavagens de 180 ul adicionais de fixador. NOTA: É importante para misturar completamente os embriões durante as lavagens por pipetagem suave para cima e para baixo várias vezes. Não fazer isso pode reduzir a relação sinal-ruído. </li><li> Deixar os embriões para fixar na segunda lavagem fixador durante 50 min a 1 h à temperatura ambiente (TA) em paraformaldeído grau de microscopia electrónica de 4% que consiste em MOPS 10 mM, pH 7,0, 0,1% de Tween-20, e artificial seawateR (ASW). Faça esta solução fresca de cada vez para obter melhores resultados. Além disso, para facilidade e praticidade embriões foram fixados em placas de 96 poços. <ol><li> Para 20 mL de utilização fixador 5 mL de 16% de paraformaldeído, 15 mL ASW, 200 mL 1 M de MOPS pH 7,0, e 20 uL de Tween-20. </li></ol></li><li> Lavar 5 vezes à temperatura ambiente com 180 ul de MOPS tampão de lavagem consistindo em MOPS 0,1 M pH 7,0, NaCl 0,5 M e 0,1% de Tween-20 durante pelo menos 5 min ou até que os embriões cair para o fundo do poço. Mais uma vez, é importante para misturar bem os embriões para o tampão de lavagem cuidado pipetando para cima e para baixo várias vezes. MOPS tampão de lavagem pode ser utilizado para 2 dias se armazenado a 4 ° C. <ol><li> Para 40 mL de MOPS lavar utilização tampão 4 ml de MOPS 1 M pH 7,0, 4 mL de 5 M de NaCl, 32 mL de dH2O e 40 ul de Tween-20. </li><li> Embriões fixos pode ser armazenado a 4 ° C durante 2 dias. NOTA: Se o armazenamento de embriões fixos mais, em seguida, adicionar 0,2% de azida de sódio em MOPS tampão de lavagem para impedir o crescimento bacteriano.</li></ol></li></ol></li><li> Pré-hibridação <ol><li> Aspirar as MOPS tampão de lavagem e adicionar 180 uL de uma proporção de 2: 1 de tampão de lavagem para MOPS tampão de hibridação, mistura embriões para dentro da solução por pipetagem suave várias vezes, e incuba-se durante pelo menos 20 min à temperatura ambiente. tampão de hibridação constituída por formamida a 70%, MOPS 0,1 M pH 7,0, NaCl 0,5 M, 1 mg / mL de BSA e 0,1% de Tween-20. <ol><li> Para 40 mL de tampão de hibridação utilização 4 ml de MOPS 1 M pH 7,0, 4 mL de NaCl 5 M, 4 mL de dH 2 O, 0,04 g de BSA, e 40 uL de Tween-20. Misture bem num vórtice, e adicionar 28 ml de formamida, e novamente vórtice. </li></ol></li><li> Remover a relação de 2: 1 de MOPS lavar e tampões de hibridação e adicionar 180 uL de uma proporção de 1: 2 de MOPS tampão de lavagem de tampão de hibridação, mistura embriões para dentro da solução, e incuba-se durante pelo menos 20 min à temperatura ambiente. </li><li> Antes de hibridação com sonda de misturar suavemente embriões em 100 - 150 ul de tampão de hibridação. Incubar os embriões a 50 ° C durante pelo menos 1h. NOTA: A incubação de embriões durante a noite é aceitável. Antes da incubação, os poços de selar com uma folha adesiva, a fim de evitar a evaporação. </li></ol></li><li> hibridização <ol><li> Num tubo separado adicionar 0,1-0,3 ng / mL de sonda e 500 ug / mL de ARNt de levedura em tampão de hibridação, seguida de vórtice suavemente para criar solução de sonda. ARNt de levedura é adicionada à solução de sonda para diminuir a ligação não específica da sonda anti-sentido. Pré-aqueça esta solução a 50 ° C e tampão de hibridação aspirado. </li><li> Misture embriões pré-hibridados em 100 mL da solução de sonda, selar com uma folha adesiva, e hibridar a 50 ° C durante 2 a 7 dias dependendo da sonda (sonda foxq2 podem ser incubadas durante 2 dias). NOTA: Os tempos de incubação varia com base fora da sonda. Alguns genes expressos em níveis baixos requerer até um período de incubação de 7 dias. placas de 96 poços pode ser colocado em uma caixa de umidade como um seguro contra a evaporação se os ADHESfolha ive não selar corretamente. </li><li> Lavagem de 5 vezes a 50 ° C com 180 ul de MOPS recém-preparadas tampão de lavagem para um total de 3 h. Em seguida, lavar 3 vezes (15 min) com 180 uL de tampão de MOPS lavar à TA. Lembre-se de misturar os embriões para o tampão de lavagem a cada vez com cuidado pipetando várias vezes. </li></ol></li><li> incubação de anticorpos <ol><li> Aspirar as MOPS tampão de lavagem e misturar embriões em 180 uL de tampão de bloqueio que consiste em 10% soro de ovelha normal e 5 mg / mL de BSA em MOPS tampão de lavagem e incubar durante pelo menos 45 min à temperatura ambiente ou a 4 ° C durante a noite. </li><li> Remover o tampão de bloqueio e, em seguida, misturar embriões em tampão contendo um bloqueio 1: 1500 de diluição de anticorpo anti-digoxigenina conjugado com fosfatase alcalina diluído em tampão de bloqueio. Incubar durante a noite à temperatura ambiente numa placa selada para evitar a evaporação. NOTA: Não deixe de anticorpos em mais do que durante a noite. </li><li> Lavar embriões 6 vezes (5 min ou até que os embriões gota) em MOPS tampão de lavagem à temperatura ambiente. embriões podemser armazenado durante a noite a 4 ° C. </li></ol></li><li> No desenvolvimento in situ <ol><li> Lavar os embriões 3 vezes (10 min) à temperatura ambiente com tampão acabado de fazer pH 9,5 constituído por Tris 0,1 M pH 9,5, NaCl 100 mM, MgCl2 50 mM, Levamisol 1 mM e 0,1% de Tween-20. <ol><li> Para 20 mL de tampão de pH 9,5 utilização 2 mL de 1 M de Tris pH 9,5, 400 ul de NaCl 5 M, 1 mL de 1 M de MgCl2, 20 uL de Tween-20, 16,6 ml de dH2O, e 0,0048 g Levamisol. </li></ol></li><li> Incubar os embriões a 37 ° C em tampão de coloração protegida da luz. Tampão de Coloração consiste de 10% de dimetil formamida, 4,5 mL / mL de 4-nitro cloreto de azul de tetrazólio (NBT) e 3,5 uL / ​​mL de 5-bromo-4-cloro-3-indolil-fosfato (BCIP) em recém-feitos tampão de pH 9,5 . <ol><li> Para 1 mL de tampão de coloração utilizar 100 mL de dimetilformamida, 4,5 mL de NBT e BCIP 3,5 mL. </li></ol></li><li> Parar a reacção com fosfatase alcalina de lavagem de 3 a 5 vezes em tampão MOPS lavar. O wi reaçãoth da sonda foxq2 tipicamente leva cerca de 30 min a 1 h. Algumas sondas podem necessitar de incubação durante a noite a RT, quer ou 4 ° C. </li><li> Misture embriões em uma solução de lavagem de MOPS 70% e 30% de glicerol. O glicerol proporciona um índice de refracção necessárias para microscopia. Os embriões podem ser armazenados nesta solução por várias semanas. Selar as placas com parafina de plástico para evitar a evaporação. </li></ol></li><li> Preparação da lâmina e captura de imagem <ol><li> Prepare um slide, organizando três pequenas tiras de fita dupla face em um triângulo com pequenos intervalos entre as tiras numa lâmina. </li><li> Transferir os embriões na lavagem MOPS 70% e solução de glicerol a 30% para o centro do triângulo e cobrir com uma lamela. </li><li> Selar a lamela por aplicação de uma camada de unha polonês em torno das arestas da lamela. </li><li> Capturar imagens usando um microscópio composto luz com um objetivo 20X e uma câmera acoplada. </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+hedgehog+signaling+to+the+establishment+of+left-right+asymmetry+in+the+sea+urchin.">Contribution of hedgehog signaling to the establishment of left-right asymmetry in the sea urchin.</a> Dev Biol. 411, 314-324 (2016).</li><li>Rottinger, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+signals+guide+migration+of+mesenchymal+cells,+control+skeletal+morphogenesis+[corrected]+and+regulate+gastrulation+during+sea+urchin+development.">FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis [corrected] and regulate gastrulation during sea urchin development.</a> Development. 135, 353-365 (2008).</li><li>Warner, J. F., McCarthy, A. M., Morris, R. L., McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+requires+motile+cilia+in+the+sea+urchin.">Hedgehog signaling requires motile cilia in the sea urchin.</a> Mol Biol Evol. 31, 18-22 (2014).</li><li>Technau, U., Steele, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology.">Evolutionary crossroads in developmental biology.</a> Cnidaria. Development. 138, 1447-1458 (2011).</li><li>Yaguchi, J., Takeda, N., Inaba, K., Yaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+Wnt-Nodal+Signals+Regulate+the+Patterning+of+Anterior+Neuroectoderm.">Cooperative Wnt-Nodal Signals Regulate the Patterning of Anterior Neuroectoderm.</a> PLoS Genet. 12, e1006001 (2016).</li><li>Duboc, V., Rottinger, E., Besnardeau, L., Lepage, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+and+BMP2/4+signaling+organizes+the+oral-aboral+axis+of+the+sea+urchin+embryo.">Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo.</a> Dev Cell. 6, 397-410 (2004).</li><li>Bradham, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chordin+is+required+for+neural+but+not+axial+development+in+sea+urchin+embryos.">Chordin is required for neural but not axial development in sea urchin embryos.</a> Dev Biol. 328, 221-233 (2009).</li><li>Su, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+for+ectoderm+specification+in+sea+urchin+embryos.">Gene regulatory networks for ectoderm specification in sea urchin embryos.</a> Biochimica et biophysica acta. 1789, 261-267 (2009).</li><li>Lin, C. Y., Su, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+in+sea+urchin+embryos+by+using+a+CRISPR/Cas9+system.">Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.</a> Dev Biol. 409, 420-428 (2016).</li></ol>

The authors have nothing to disclose.

A metodologia aqui apresentada é um exemplo que ilustra o poder de usar embriões com menos complexidade genômica e morfológica de vertebrados para compreender as vias de transdução de sinalização e GRNs regem mecanismos de desenvolvimento fundamentais .. Muitos laboratórios estão usando ensaios semelhantes durante o desenvolvimento do ouriço-do-mar cedo para dissecar o vias envolvidas em outros eventos de especificação destino de sinalização celular (por exemplo, Notch, Hedgehog, TGF-β, e FGF si...

redes de genes reguladores (GRNs) e vias de transdução de sinal estabelecer a expressão espacial e temporal dos genes durante o desenvolvimento embrionário que são usados ​​para construir o plano de adulto corpo do animal. Célula-a-célula vias de transdução de sinal são componentes essenciais destas redes reguladoras, que proporciona os meios pelos quais as células comunicam. Estas interacções celulares estabelecer e aperfeiçoar a expressão de genes reguladores e diferenciação dentro e entre os vários territórios durante a embriogênese 1, 2. Interações entre os moduladores secretadas extracelulares (ligantes, antagonistas), receptores e co-receptores controlar as atividades de vias de transdução de sinal. Uma variedade de moléculas intracelulares transduz estas entradas resultando na expressão alterada do gene, divisão, e / ou a forma de uma célula. Enquanto muitas das moléculas-chave utilizadas nos níveis extracelulares e intracelulares nas vias principais sãoconhecido, é um conhecimento incompleto, devido em grande parte à complexidade das vias de sinalização individuais. Além disso, diferentes vias de sinalização, muitas vezes interagem uns com os outros, positivamente ou negativamente na extracelular, intracelular, e os níveis de transcrição de 3, 4, 5, 6. É importante ressaltar que os principais componentes de vias de transdução de sinal são altamente conservadas em todas as espécies de metazoários, e, curiosamente, a maioria das principais vias de sinalização, muitas vezes executar funções de desenvolvimento semelhantes em muitas espécies quando se comparam os organismos de filos intimamente relacionados, em especial, 7, 8, 9, 10, 11. O estudo da sinalização durante o desenvolvimento é uma tarefa difícil em qualquer organismo, e hávários desafios significativos para estudar vias de sinalização na maioria dos modelos deuterostomia (vertebrados, cordados invertebrados, hemichordates, e equinodermes): 1) Em vertebrados há um grande número de possíveis ligandos e as interacções receptor / co-moduladores, moléculas de transdução intracelular, bem como potenciais interacções entre diferentes vias de sinalização, devido à complexidade do genoma de 12, 13, 14; 2) A morfologia complexa e movimentos morfogenéticos em vertebrados muitas vezes tornam mais difíceis de interpretar as interações funcionais em e entre vias de transdução de sinal; 3) As análises na maioria dos não-equinodermos espécie modelo de invertebrados deuterostomia são limitados por janelas curtas de gravidity com exceção de algumas espécies de tunicados 15, 16. oouriço-do-mar embrião tem algumas das limitações acima mencionadas e oferece muitas qualidades únicas para a realização de uma análise detalhada das vias de transdução de sinal in vivo. Estes incluem o seguinte: 1) A relativa simplicidade do genoma do ouriço-do-mar reduz significativamente o número de possíveis do ligando, do receptor / co-receptor e molécula de transdução intracelular interacções 17; 2) Os GRNs que controlam a memória descritiva e modelação das camadas germinativas e grandes eixos embrionários são bem estabelecida em embriões de ouriço-do-mar, auxiliando na compreensão do contexto regulamentar da célula / território receber os sinais 18, 19; 3) Muitas vias de transdução de sinal podem ser estudadas entre as fases de clivagem e de gástrula inicial quando embriões consistem de um único epitélio em camadas cuja morfologia é mais fácil de analisar; 4) As moléculas envolvemd nas vias de sinalização nas ouriços do mar são facilmente manipulados; 5) Muitas ouriços do mar estão grávidas de 10 a 11 meses por ano (por exemplo, Strongylocentrotus purpuratus e Lytechinus variegatus). Aqui, apresentamos um método para caracterizar de forma sistemática e eficiente componentes das vias de sinalização que especificam e territórios padrão em embriões de ouriço-do-mar para ilustrar as vantagens que vários sistemas modelo de invertebrados oferecem no estudo dos mecanismos moleculares complexas.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Translational-blocking morpholino and/or splice-blocking morpholino</td>
			<td>Gene Tools LLC</td>
			<td>Customized</td>
			<td>More information at www.gene-tools.com</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Invitrogen</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC (dextran fluorescein isothiocyanate)</td>
			<td>Invitrogen, Life Technologies</td>
			<td>D1821</td>
			<td>Make 25mg/mL stock solution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution EM Grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma Aldrich</td>
			<td>M1254-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>23336-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma Aldrich</td>
			<td>47671-1L-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast tRNA</td>
			<td>Invitrogen</td>
			<td>15401-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase-conjugated anti-digoxigenin antibody</td>
			<td>Roche</td>
			<td>11 093 274 910</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramisole hydrochloride (levamisole)</td>
			<td>Sigma Aldrich</td>
			<td>L9756-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base UltraPure</td>
			<td>Research Products Internationall Corp</td>
			<td>56-40-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>7786-30-3</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP (5-Bromo-4-Chloro-3-indolyl-phosphate</td>
			<td>Roche</td>
			<td>11 383 221 001</td>
			<td></td>
		</tr>
		<tr>
			<td>4 Nitro blue tetrazolium chloride (NBT)</td>
			<td>Roche</td>
			<td>11 383 213 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Formamide</td>
			<td>Sigma Aldrich</td>
			<td>D4551-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M7506-2KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>C1016-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

the power of simplicity: sea urchin embryos as in vivo developmental models for studying complex cell-to-cell signaling network interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

No embrião ouriço-do-mar temos demonstrado que 3 diferentes sinalização Wnt ramos (Wnt / β-catenina, Wnt / JNK, e Wnt / PKC) 4, 25 interagem de modo a formar uma rede de sinalização de Wnt que governa ântero-posterior padronização (AP). Uma das consequências mais importantes destes eventos de sinalização é que a GRN inicial amplamente expressa neuroectoderma anterior (ANE) torna-se restrita a um pequeno território ...

Watch this Scientific Journal Video about The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions at JoVE.com

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Este artigo de vídeo detalha uma simples em metodologia in vivo que pode ser utilizado para caracterizar de forma eficiente e sistematicamente componentes de vias de sinalização complexas e redes reguladoras em muitos embriões de invertebrados.

O Poder da Simplicidade: Sea Urchin embriões Na Vivo Modelos de desenvolvimento para estudar células de células-complexo de sinalização de rede Interações

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

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The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Unit 1

Principles of Cell Signaling

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7.8K visualizações.  Mississippi State University.  Este artigo de vídeo detalha uma simples em metodologia in vivo que pode ser utilizado para caracterizar de forma eficiente e sistematicamente componentes de vias de sinalização complexas e redes reguladoras em muitos embriões de invertebrados. 

Vídeo: The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

Protocolos para obtenção de embriões somáticos e Zygotic para estudar a regulação do desenvolvimento embrionário precoce no Modelo de leguminosas<em&gt; Medicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

Biologia do Desenvolvimento

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Caracterização de Thymic Settling Progenitores no embrião do rato Usando<em&gt; In Vivo</em&gt; E<em&gt; In Vitro</em&gt; Os ensaios

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

Estudar Wnt Signaling Durante Padronização de conduzir Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

Induzindo completa Polyp Regeneração do aboral Physa da Anémona Starlet<em&gt; Vectensis Nematostella</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Tratamento de drogas e<em&gt; In Vivo</em&gt; Imagem de Osteoblast-osteoclastos Interações em um modelo de Medaka Peixe Osteoporose

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Estimulação da sinalização Notch no mouse osteoclastos Precursores

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of pancreatic progenitor cells that can be used for the study of and future treatment of diabetes. This article outlines a four-stage differentiation protocol designed to generate pancreatic progenitor cells from human embryonic stem cells (hESCs). This protocol can be applied to a number of human pluripotent stem cell (hPSC) lines. The approach taken to generate pancreatic progenitor cells is to differentiate hESCs to accurately model key stages of pancreatic development. This begins with the induction of the definitive endoderm, which is achieved by culturing the cells in the presence of Activin A, basic Fibroblast Growth Factor (bFGF) and CHIR990210. Further differentiation and patterning with Fibroblast Growth Factor 10 (FGF10) and Dorsomorphin generates cells resembling the posterior foregut. The addition of Retinoic Acid, NOGGIN, SANT-1 and FGF10 differentiates posterior foregut cells into cells characteristic of pancreatic endoderm. Finally, the combination of Epidermal Growth Factor (EGF), Nicotinamide and NOGGIN leads to the efficient generation of PDX1+/NKX6-1+ cells. Flow cytometry is performed to confirm the expression of specific markers at key stages of pancreatic development. The PDX1+/NKX6-1+ pancreatic progenitors at the end of stage 4 are capable of generating mature &#946; cells upon transplantation into immunodeficient mice and can be further differentiated to generate insulin-producing cells in vitro. Thus, the efficient generation of PDX1+/NKX6-1+ pancreatic progenitors, as demonstrated in this protocol, is of great importance as it provides a platform to study human pancreatic development in vitro and provides a source of cells with the potential of differentiating to &#946; cells that could eventually be used for the treatment of diabetes.

Efficient Differentiation of Pluripotent Stem Cells to NKX6-1+ Pancreatic Progenitors

Here we describe a 4-stage protocol to differentiate human embryonic stem cells to NKX6-1+ pancreatic progenitors in vitro. This protocol can be applied to a variety of human pluripotent stem cell lines.

Diferenciação eficiente dos Pluripotent Stem Cells para NKX6-1<sup&gt; +</sup&gt; Progenitores pancreáticas

Pluripotent stem cells have the ability to self renew and differentiate to multiple lineages, making them an attractive source for the generation of ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

In vivo de imagens de Gene Transgenic Expressão em Individual Retinal Progenitores em embriões Zebrafish Quimérico para estudar celular Influências não-autônomos

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

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 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 ...

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