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. Stratégie de conception morpholino Throughput haut <ol><li> Identifier un gène (s) d&#39;intérêt (par exemple , l&#39; approche de gène candidat, l&#39; analyse des cis-régulatrices, et RNA-seq ou écrans différentiels / protéomique). </li><li> Utilisez génomique, transcriptomique, et des données d&#39;expression génique disponibles sur les sites Web fréquemment mis à jour (par exemple SpBase http://www.echinobase.org 20 et S. purpuratus Genome Recherche http: ///urchin.nidcr.nih.gov/blast/index .html) pour déterminer que le profil d&#39;expression spatiotemporelle chevauche avec le mécanisme de développement en question. Si aucune donnée d&#39;expression est disponible, puis générer des amorces qPCR et / ou un antisens sonde in situ pour évaluer le profil d&#39;expression génique. </li><li> Après avoir déterminé le modèle spatial et temporel d&#39;expression, d&#39;obtenir la séquence d&#39;ADN à partir des sites web génomiques. <ol><li> Pour générer des oligonucléotides morpholino traduction-bloquant, d&#39;obtenir la séquence of la «région non traduite (5 &#39;5 UTR) directement en amont du codon d&#39;initiation de la balise de séquence exprimée (EST) des bases de données disponibles sur SpBase ou S. purpuratus Genome recherche des sites Web. Si cette information est indisponible pour le gène d&#39;intérêt, puis effectuer 5 &#39;RACE. </li><li> Pour concevoir un morpholino d&#39;épissure de blocage, la recherche de la séquence génomique comprenant les exons et les introns en utilisant l&#39;outil échafaudage blastn dans SpBase ou S. purpuratus Genome outil de recherche. </li></ol></li><li> Utilisez le site Web oligonucléotide conception http://oligodesign.gene-tools.com afin de concevoir la séquence paire morpholino 25 de base désirée. <ol><li> Pour une translation morpholino-blocage, en utilisant la séquence d&#39;ARNm de 5 &#39;à 3&#39; comme source de la séquence cible de la traduction. Inclure la séquence 5 &#39;UTR (environ 70 nucléotides en amont du site de début de transcription) plus de 25 bases de la région (en aval du site de départ) de codage avec le codon de départ wi marquéème parenthèse (ATG). </li><li> Pour un morpholino d&#39;épissure de blocage, sélectionnez intron-exon ou d&#39;une séquence de frontière exon-intron et comprennent 50 bases (25 bases de séquence exon et 25 bases de séquence d&#39;introns) autour des régions limitrophes. Scanner le génome avec la séquence morpholino pour vérifier que la séquence soit unique. </li></ol></li></ol> 2. La micro-injection de morpholino oligonucleotides <ol><li> Préparer 3 mM solutions des oligonucléotides morpholino en ajoutant 100 pi d&#39;eau sans nucléase dans le 300 nmol morpholino flacon. NOTE: Ne pas utiliser l&#39;eau traitée DEPC pour la remise en suspension, car le pyrocarbonate de diéthyle peut endommager le morpholino. </li><li> Pour la première rotation de reconstitution sur le flacon contenant la solution mère d&#39;oligonucléotide pendant 30 s à pleine vitesse (14.000 - 16.000 xg), vortex brièvement, de la chaleur à 65 ° C pendant 5 à 10 min, vortex brièvement, et de garder le morpholino stock à la chambre température pendant au moins 1 h. Morpholino solution stock<html> s sont stockés de -20 ° C à +4 ° C. </li><li> Préparer une solution injectable contenant oligonucleotides morpholino, à la concentration souhaitée. Cette solution contient habituellement 20% de glycérol ou de KCl 125 en tant que support et 15% FITC-Dextran (isothiocyanate de fluorescéine dextrane 10.000 MW 2,5 mg / uL solution mère). FITC-Dextran et d&#39;autres conjugués dextrane fluorescents sont utilisés en routine pour identifier les embryons injectés par microscopie à épifluorescence. Magasin solutions d&#39;injection à -20 ° C. </li><li> Chauffer la solution morpholino à 65 ° C dans un bloc chauffant ou un bain d&#39;eau pendant au moins 2 - 5 min. </li><li> En bref tourner la solution morpholino pendant 30 s à pleine vitesse (14.000 - 16.000 xg), vortex pendant 1 min et centrifuger à pleine vitesse (14.000 - 16.000 xg) pendant au moins 10 min. </li><li> Charger les aiguilles d&#39;injection avec la solution morpholino. Pour un protocole de microinjection détaillée s&#39;il vous plaît voir Stepicheva et Song 2014 21 et Acclamations et Ettensohn 2004&quot;&gt; 22. </li></ol> 3. Fixation et In Situ Protocole à 24 h post-fécondation (HPF) dans S. purpuratus Embryons NOTE: Ce protocole est modifié de Arenas-Mena et al. 2000 23 et Sethi et al. 2014 24. <ol><li> Fixation <ol><li> Ajouter quelques gouttes de fixateur (voir ci-dessous) à des puits contenant des embryons, mélanger doucement par pipetage, et leur permettre de se déposer. Retirer la solution de fixateur, puis mélanger les embryons avec deux lavages supplémentaires 180 uL de fixateur. NOTE: Il est important de bien mélanger les embryons pendant les lavages par pipetage doux monter et descendre plusieurs fois. Ne pas le faire peut réduire le rapport signal sur bruit. </li><li> Laissez les embryons à fixer dans le second lavage de fixateur pendant 50 min à 1 h à température ambiante (RT) dans 4% de paraformaldehyde de qualité en microscopie électronique consistant en mM MOPS 10 pH 7,0, 0,1% de Tween-20, et artificielle seawater (ASW). Faire de cette solution fraîche chaque fois pour de meilleurs résultats. En outre, pour la facilité et l&#39;aspect pratique des embryons sont fixés dans des plaques à 96 puits. <ol><li> Pour 20 ml de fixatif utiliser 5 ml 16% de paraformaldehyde, 15 ml ASW, 200 pi de 1 M de MOPS à pH 7,0, et 20 pi de Tween-20. </li></ol></li><li> Laver 5 fois à température ambiante avec 180 pi de tampon MOPS lavage consistant en 0,1 M de MOPS à pH 7,0, 0,5 M de NaCl et 0,1% de Tween-20 pendant au moins 5 minutes, ou jusqu&#39;à ce que des embryons tomber au fond du puits. Encore une fois, il est important de bien mélanger les embryons dans le tampon de lavage par pipetage doucement monter et descendre plusieurs fois. tampon de lavage MOPS peut être utilisé pendant 2 jours si elle est conservée à 4 ° C. <ol><li> 40 ml de MOPS tampon de lavage utilisé 4 ml de MOPS 1 M pH 7,0, 4 ml de NaCl 5 M, 32 ml dH 2 O, et 40 pi de Tween-20. </li><li> Embryons fixes peuvent être conservés à 4 ° C pendant 2 jours. NOTE: Si le stockage des embryons fixes plus, puis ajouter de l&#39;azoture de sodium à 0,2% en MOPS tampon de lavage pour empêcher la croissance bactérienne.</li></ol></li></ol></li><li> Préhybridation <ol><li> Aspirer le MOPS tampon de lavage et ajouter 180 ul d&#39;un rapport de 2: 1 de MOPS tampon de lavage au tampon d&#39;hybridation, mélanger les embryons dans la solution par pipetage doux à plusieurs reprises, et incuber pendant au moins 20 min à température ambiante. un tampon d&#39;hybridation est constitué de 70% de formamide, 0,1 M de MOPS à pH 7,0, 0,5 M de NaCl, 1 mg / ml de BSA et 0,1% de Tween-20. <ol><li> Pour 40 mL utilisation du tampon d&#39;hybridation 4 ml 1 M MOPS pH 7,0, 4 ml de NaCl 5 M, 4 ml dH 2 O, 0,04 g de BSA, et 40 pi de Tween-20. Bien mélanger par tourbillonnement, ajouter 28 ml de formamide, et le vortex à nouveau. </li></ol></li><li> Retirez le rapport 2: 1 de MOPS laver et les tampons d&#39;hybridation et ajouter 180 ul d&#39;un rapport 1: 2 de MOPS tampon de lavage au tampon d&#39;hybridation, mélanger les embryons dans la solution, et incuber pendant au moins 20 min à température ambiante. </li><li> Avant hybridation de sonde mélanger délicatement les embryons dans 100 - 150 pi de tampon d&#39;hybridation. Incuber les embryons à 50 ° C pendant au moins 1h. NOTE: L&#39;incubation des embryons nuit est acceptable. Avant l&#39;incubation, sceller les puits avec une feuille adhésive afin d&#39;éviter l&#39;évaporation. </li></ol></li><li> Hybridation <ol><li> Dans un tube séparé ajouter 0,1 à 0,3 ng / pl de sonde et 500 pg / ml d&#39;ARNt de levure dans un tampon d&#39;hybridation, puis doucement vortex pour créer une solution de sonde. ARNt de levure est ajoutée à la solution de la sonde pour réduire la liaison non spécifique de la sonde anti-sens. Préchauffer cette solution à 50 ° C et le tampon d&#39;hybridation aspirat. </li><li> Mélanger les embryons pré-hybridés dans 100 ul de la solution de sonde, sceller avec une feuille adhésive, et hybrident à 50 ° C pendant 2 à 7 jours en fonction de la sonde (la sonde foxq2 peut être mis à incuber pendant 2 jours). NOTE: Les temps d&#39;incubation peuvent varier en fonction de la sonde. Certains gènes exprimés à des niveaux faibles nécessitent jusqu&#39;à une incubation de 7 jours. plaques à 96 puits peuvent être placés dans une boîte d&#39;humidité comme une assurance contre l&#39;évaporation si les ADHESfeuille ive ne parvient pas à sceller correctement. </li><li> Laver 5 fois à 50 ° C avec 180 pi de MOPS fraîchement préparé le tampon de lavage pour un total de 3 h. Ensuite, laver 3 fois (15 min) avec 180 pi de MOPS tampon de lavage à température ambiante. Rappelez-vous de mélanger les embryons dans le tampon de lavage à chaque fois par un léger pipetage plusieurs fois. </li></ol></li><li> Anticorps incubation <ol><li> Aspirer le MOPS tampon de lavage et mélanger embryons dans 180 pi de tampon de blocage constitué de 10% sérum normal de mouton et 5 mg / ml de BSA dans MOPS tampon de lavage et incuber pendant au moins 45 min à température ambiante ou à 4 ° C pendant la nuit. </li><li> Retirer le tampon de blocage, puis mélanger les embryons dans un tampon de blocage contenant une dilution à 1: 1500 d&#39;anticorps anti-digoxigénine conjugué à la phosphatase alcaline diluée dans un tampon de blocage. Incuber une nuit à température ambiante dans une plaque scellée pour éviter l&#39;évaporation. NOTE: Ne laissez pas l&#39;anticorps sur plus d&#39;un jour. </li><li> Laver les embryons 6 fois (5 min ou jusqu&#39;à ce que les embryons tombent) dans MOPS tampon de lavage à température ambiante. embryons peutêtre stocké pendant une nuit à 4 ° C. </li></ol></li><li> Dans le développement in situ <ol><li> Laver les embryons 3 fois (10 min) à température ambiante avec fraîchement préparée , pH 9,5 tampon constitué de Tris 0,1 M pH 9,5, 100 mM de NaCl, 50 mM de MgCl2, 1 mM de lévamisole, et du Tween-20 à 0,1%. <ol><li> Pour 20 ml de tampon pH 9,5 utiliser 2 ml de Tris 1 M pH 9,5, 400 ul de NaCl 5 M, 1 ml de 1 M de MgCl2, 20 pi de Tween-20, 16,6 ml dH 2 O et 0,0048 g lévamisole. </li></ol></li><li> Incuber les embryons à 37 ° C dans un tampon de coloration à l&#39;abri de la lumière. un tampon de coloration est constitué de 10% de dimethyl formamide, 4,5 ul / ml de 4-nitro tétrazolium (NBT) et 3,5 ul / ml de 5-bromo-4-chloro-3-indolyl-phosphate (BCIP) dans fraîchement préparé le tampon à pH 9,5 . <ol><li> Pour 1 ml de tampon de coloration en utilisant 100 ul dimethylformamide, 4,5 ul de NBT et 3,5 ul BCIP. </li></ol></li><li> Arrêter la réaction de la phosphatase alcaline par lavage 3 à 5 fois dans MOPS tampon de lavage. La réaction wie la sonde foxq2 prend généralement 30 min à 1 h. Certaines sondes peuvent avoir besoin d&#39;une nuit d&#39;incubation soit à la température ambiante ou à 4 ° C. </li><li> Mélanger les embryons dans une solution de 70% de lavage MOPS et 30% de glycérol. Le glycérol fournit un indice de réfraction nécessaire pour la microscopie. Les embryons peuvent être conservés dans cette solution pendant plusieurs semaines. Sceller les plaques avec de la paraffine en plastique pour éviter l&#39;évaporation. </li></ol></li><li> Préparation de diapositives et de capture d&#39; image <ol><li> Préparer une diapositive en organisant trois petites bandes de ruban adhésif double face dans un triangle avec de petits écarts entre les bandes sur une lame. </li><li> Transférer les embryons dans le lavage de MOPS 70% et une solution de glycérol à 30% au centre du triangle et couvrir avec une lamelle. </li><li> Sceller la lamelle en appliquant une couche de vernis à ongles sur les bords de la lamelle. </li><li> Capture d&#39;images à l&#39;aide d&#39;un microscope optique composé avec un objectif 20X et une caméra attachée. </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.

La méthodologie présentée ici est un exemple qui illustre le pouvoir d&#39;utiliser des embryons avec une complexité moins génomique et morphologique que les vertébrés à comprendre les voies de signalisation de la transduction et GRNS régissant les mécanismes de développement fondamentaux .. De nombreux laboratoires utilisent des tests similaires au début du développement de l&#39;oursin à disséquer le voies impliquées dans d&#39; autres événements de spécification du destin cellulaire de signalisatio...

réseaux de gènes de régulation (GRNS) et voies de signalisation établissent l&#39;expression spatiale et temporelle des gènes au cours du développement embryonnaire qui sont utilisés pour construire le plan corporel de l&#39;animal adulte. Cell-à-cellule des voies de transduction du signal sont des composants essentiels de ces réseaux de régulation, en fournissant les moyens par lesquels les cellules communiquent. Ces interactions cellulaires établir et affiner l&#39;expression des gènes de régulation et de différenciation dans et entre les différents territoires pendant l&#39; embryogenèse 1, 2. Les interactions entre les modulateurs sécrétées extracellulaires (ligands, des antagonistes), des récepteurs et co-récepteurs contrôlent les activités des voies de transduction du signal. Un assortiment de molécules intracellulaires transduction ces entrées résultant de l&#39;expression altérée du gène, la division et / ou la forme d&#39;une cellule. Bien que plusieurs des molécules clés utilisés aux niveaux extracellulaires et intracellulaires dans les principales voies sontconnu, il est une connaissance incomplète due en grande partie à la complexité des voies de signalisation individuelles. En outre, les différentes voies de signalisation interagissent souvent entre eux de façon positive ou négative au extracellulaire, intracellulaire, et les niveaux de transcription 3, 4, 5, 6. Fait important, les composants de base de voies de signalisation sont hautement conservées dans toutes les espèces de métazoaires, et, fait remarquable, la plupart des grandes voies de signalisation exercent souvent des fonctions de développement similaires dans de nombreuses espèces lorsque l&#39;on compare les organismes de phyla étroitement liés , en particulier 7, 8, 9, 10, 11. L&#39;étude de la signalisation au cours du développement est une tâche ardue dans tout organisme, et il yPlusieurs défis importants pour l&#39; étude des voies de signalisation dans la plupart des modèles deutérostomes (vertébrés, chordés invertébrés, hémichordés et échinodermes): 1) Chez les vertébrés , il y a un grand nombre de possibles ligand et les interactions récepteur / co-modulateurs, des molécules de transduction intracellulaire, ainsi que les interactions possibles entre les différentes voies de signalisation en raison de la complexité du génome 12, 13, 14; 2) La morphologie complexe et mouvements morphogénétiques chez les vertébrés font qu&#39;il est souvent plus difficile d&#39;interpréter les interactions fonctionnelles et entre les voies de transduction du signal; 3) Les analyses de la plupart des espèces modèles invertébré deutérostomien non échinodermes sont limitées par de courtes fenêtres de gravidity à l&#39;exception de certaines espèces de tuniciers 15, 16. leoursin embryon a quelques - unes des limitations mentionnées ci-dessus et offre de nombreuses qualités uniques pour effectuer une analyse détaillée des voies de transduction du signal in vivo. Ceux - ci sont les suivantes: 1) La relative simplicité du génome de l&#39; oursin réduit de manière significative le nombre de possible ligand, un récepteur / co-récepteur et la molécule de transduction intracellulaire interactions 17; 2) Les GRNS contrôlant la spécification et la structuration des couches germinales et les grands axes embryonnaires sont bien établis dans les embryons d&#39;oursins, d&#39; aider à la compréhension du contexte réglementaire de la cellule / territoire recevant les signaux 18, 19; 3) De nombreuses voies de transduction du signal peuvent être étudiés entre les étapes de clivage et gastrula lorsque les embryons sont constitués d&#39;un seul épithélium stratifié dont la morphologie est plus facile à analyser; 4) Les molécules comportentd dans les voies dans les oursins sont facilement manipulables de signalisation; 5) De nombreux oursins sont gravides pendant 10 à 11 mois par an (par exemple Strongylocentrotus purpuratus et Lytechinus variegatus). Ici, nous présentons une méthode pour caractériser systématiquement et efficacement les composants des voies de signalisation qui précisent et territoires de modèle dans les embryons d&#39;oursins pour illustrer les avantages que plusieurs systèmes de modèles d&#39;invertébrés offrent dans l&#39;étude des mécanismes moléculaires complexes.

<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.

Dans l&#39;embryon d&#39;oursin , nous avons montré que 3 différents de signalisation Wnt branches (Wnt / β-caténine, Wnt / JNK et Wnt / PKC) 4, 25 interagissent pour former un réseau de signalisation Wnt qui régit antéro-postérieur (AP) patterning. Une des conséquences les plus importantes de ces événements de signalisation est que la large exprimé neurectoderme antérieure (ANE) GRN initial devient restreint à un p...

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

Cet article vidéo détaille un simple dans la méthode in vivo qui peut être utilisé pour caractériser systématiquement et efficacement les composants de voies de signalisation complexes et les réseaux de régulation dans de nombreux embryons d&#39; invertébrés.

La puissance de la simplicité: Sea Urchin Embryons comme<em&gt; In Vivo</em&gt; Modèles de développement pour l&#39;étude de la cellule cellule-à-Complex de signalisation réseau Interactions

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

The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the signaling pathways and the gene regulatory networks that they activate in an in vivo developmental context.Demonstrating this procedure will be Marina Martinez-Bartolom

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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 vues.  Mississippi State University. The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the si...

Vidéo: 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.

Protocoles pour l&#39;obtention Zygotic et embryons somatiques pour l&#39;étude de la réglementation de développement précoce de l&#39;embryon dans la légumineuse modèle<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 ...

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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.

Caractérisation des progéniteurs thymique décantation dans l&#39;embryon de souris Utilisation<em&gt; In Vivo</em&gt; Et<em&gt; In Vitro</em&gt; Essais

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.

Étude Wnt Signaling Pendant Patterning de Conduite 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.

Induire complète Polyp Régénération du aboral Physa de la mer Anémone Starlet<em&gt; Nematostella vectensis</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.

Traitement de la toxicomanie et<em&gt; In Vivo</em&gt; Imagerie des ostéoblastes-ostéoclastes Interactions dans un modèle Medaka poisson Ostéoporose

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

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.

Imagerie in vivo de Transgenic Gene Expression en Retinal individuelle progéniteurs dans chimériques Zebrafish Embryons à étudier cellulaires Influences non autonomes

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.

Imagerie en direct de la souris Fusion de palais secondaire

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).

Visualiser le nœud et la notochorde plaque en Gastrulating des embryons de souris à l’aide de la microscopie électronique à balayage et toute monture Immunofluorescence

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

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

Évaluation des dommages oxydatifs dans les cellules de surface oculaires de la souris primaire/cellules souches en réponse aux dommages causés par les ultraviolets-C (UV-C)

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Une méthode de montage en couches pour la microscopie confocale de temps-lapse prolongée des embryons entiers de poisson zèbre

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...