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. Strategia High Throughput Morpholino design <ol><li> Identificare un gene (s) di interesse (ad esempio, candidato approccio del gene, l&#39;analisi cis-normativo, RNA-Seq e / o schermi differenziali proteomica). </li><li> Utilizzare genomica, trascrittomica, e dati di espressione genica disponibili sui siti web aggiornati di frequente (ad es SpBase http://www.echinobase.org 20 e S. purpuratus Genome Ricerca http: ///urchin.nidcr.nih.gov/blast/index .html) per determinare il profilo di espressione spazio-temporale si sovrappone con il meccanismo di sviluppo in questione. Se non ci sono dati di espressione è disponibile, quindi generare primer qPCR e / o un antisenso sonda situ per valutare il pattern di espressione genica. </li><li> Dopo aver determinato il pattern di espressione spaziale e temporale, ottenere la sequenza del DNA dai siti web genomici. <ol><li> Per la generazione di oligonucleotidi morfolino traduzione di blocco, è necessario ottenere la sequenza of la &#39;regione non tradotti (5&#39; 5 UTR) direttamente a monte del codone iniziazione da expressed sequence tag (EST) banche dati disponibili sul SpBase o S. purpuratus Genome di ricerca siti web. Se questa informazione non è disponibile per il gene di interesse, quindi eseguire 5 &#39;RACE. </li><li> Per progettare un morfolino giunzione di blocco, la ricerca della sequenza genomica compresi gli esoni e introni utilizzando lo strumento patibolo BLASTN in SpBase o S. purpuratus Genome strumento di ricerca. </li></ol></li><li> Utilizzare il sito oligonucleotide progettazione http://oligodesign.gene-tools.com al fine di progettare la base di 25 sequenza di coppia morfolino desiderato. <ol><li> Per un morfolino traslazionale-blocco, utilizzare la sequenza di mRNA da 5 &#39;a 3&#39; come sorgente di traslazione sequenza bersaglio. Includere il 5 &#39;UTR sequenze (circa 70 nucleotidi a monte del sito di inizio della trascrizione) più 25 basi di regione codificante (a valle del sito di inizio) con l&#39;inizio codone marcato wiesima parentesi (ATG). </li><li> Per un morfolino giunzione di blocco, selezionare introne-esone o esone-introne sequenza di confine e comprendono 50 basi (25 basi della sequenza dell&#39;esone e 25 basi di sequenze introne) attorno alle regioni di confine. Scansione genoma con la sequenza morfolino per verificare che la sequenza è unica. </li></ol></li></ol> 2. Microiniezione di oligonucleotidi Morpholino <ol><li> Preparare 3 soluzioni madre mm di oligonucleotidi morfolino con l&#39;aggiunta di 100 ml di acqua priva di nucleasi nel morfolino flacone 300 nmol. NOTA: Non usare acqua trattata DEPC per la risospensione perché dietilpirocarbonato può danneggiare il morfolino. </li><li> Per la prima rotazione ricostituzione lungo il flaconcino contenente la soluzione di riserva oligonucleotide per 30 s alla massima velocità (14.000 - 16.000 xg), vortex brevemente, il calore a 65 ° C per 5 a 10 minuti, vortex brevemente, e mantenere lo stock morfolino in camera temperatura per almeno 1 h. Soluzione morfolino<html> s vengono memorizzati da -20 ° C a + 4 ° C. </li><li> Preparare la soluzione iniettabile contenente oligonucleotidi morfolino alla concentrazione desiderata. Questa soluzione di solito contiene il 20% di glicerolo o 125 mm KCl come vettore e il 15% FITC-destrano (fluoresceina isotiocianato destrano 10.000 MW 2,5 mg / ml di soluzione). FITC-destrano e altri coniugati destrano fluorescenti sono abitualmente utilizzati per identificare gli embrioni iniettati mediante microscopia in epifluorescenza. Le soluzioni di iniezione conservare a -20 ° C. </li><li> Riscaldare la soluzione morfolino a 65 ° C in un blocco di calore oa bagnomaria per almeno 2 - 5 min. </li><li> Brevemente girare la soluzione morfolino per 30 s alla massima velocità (14.000 - 16.000 xg), vortex per 1 minuto e centrifugare a massima velocità (14.000 - 16.000 xg) per almeno 10 minuti. </li><li> Caricare i aghi da iniezione con la soluzione morfolino. Per una dettagliata protocollo microiniezione vedere Stepicheva e Song 2014 21 e applausi e Ettensohn 2004&quot;&gt; 22. </li></ol> 3. Fissazione e in situ protocollo a 24 ore post-fecondazione (HPF) a S. purpuratus Embrioni NOTA: Questo protocollo viene modificato da Arenas-Mena et al. 2000 23 e Sethi et al. 2014 24. <ol><li> Fissazione <ol><li> Aggiungere qualche goccia di fissativo (vedi sotto) per pozzetti contenenti gli embrioni, mescolare delicatamente pipettando, e permettere loro di stabilirsi. Rimuovere la soluzione fissativo, e poi mescolare gli embrioni con due ulteriori lavaggi 180 ml di fissativo. NOTA: È importante omogeneizzare gli embrioni durante i lavaggi di dolce pipettando su e giù diverse volte. In caso contrario si può abbassare il rapporto segnale-rumore. </li><li> Lasciare gli embrioni di fissare nella secondo lavaggio fissativo per 50 minuti a 1 ora a temperatura ambiente (RT) in 4% paraformaldeide microscopia elettronica grade costituito da 10 mM MOPS pH 7,0, 0,1% Tween-20, ed artificiale seawater (ASW). Rendere questa soluzione fresca ogni volta per ottenere i migliori risultati. Inoltre, per la facilità e praticità embrioni sono fissati in piastre da 96 pozzetti. <ol><li> Per 20 mL uso fissativo 5 ml 16% paraformaldeide, 15 mL ASW, 200 ml 1 M MOPS pH 7,0, e 20 ml di Tween-20. </li></ol></li><li> Lavare 5 volte a RT con 180 ml di MOPS lavare tampone costituito da 0,1 M MOPS pH 7,0, 0,5 M NaCl e 0,1% Tween-20 per almeno 5 minuti o fino embrioni cadere sul fondo del pozzo. Anche in questo caso, è importante mescolare bene gli embrioni nel tampone di lavaggio pipettando gentilmente su e giù parecchie volte. Il tampone di lavaggio MOPS può essere utilizzato per 2 giorni se conservato a 4 ° C. <ol><li> Per 40 mL MOPS lavano uso di buffer da 4 ml 1 M MOPS pH 7,0, 4 ml 5 M di NaCl, 32 mL dH 2 O, e 40 ml di Tween-20. </li><li> Embrioni fissi possono essere conservati a 4 ° C per 2 giorni. NOTA: Se la memorizzazione di embrioni fisse più, quindi aggiungere lo 0,2% di sodio azide in MOPS tampone di lavaggio per prevenire la crescita batterica.</li></ol></li></ol></li><li> Pre-ibridazione <ol><li> Aspirare il MOPS tampone di lavaggio e aggiungere 180 ml di un rapporto di 2: 1 di MOPS tampone di lavaggio in tampone di ibridazione, mescolare embrioni nella soluzione delicatamente pipettaggio più volte, e incubare per almeno 20 minuti a temperatura ambiente. tampone di ibridazione consiste di 70% formammide, 0,1 M pH 7,0 MOPS, 0,5 M NaCl, 1 mg / ml BSA e 0,1% Tween-20. <ol><li> Per 40 mL uso tampone di ibridazione 4 ml 1 M MOPS pH 7,0, 4 ml 5 M di NaCl, 4 mL dH 2 O, 0,04 g BSA, e 40 ml di Tween-20. Mescolare bene nel vortex, aggiungere 28 ml di formammide, e vortex di nuovo. </li></ol></li><li> Rimuovere il rapporto 2: 1 di MOPS lavare e buffer di ibridazione e aggiungere 180 ml di un rapporto 1: 2 del MOPS tampone di lavaggio in tampone di ibridazione, mescolare embrioni nella soluzione, e incubare per almeno 20 minuti a temperatura ambiente. </li><li> Prima sonda di ibridazione mescolare delicatamente embrioni in 100 - 150 ml di buffer di ibridazione. Incubare embrioni a 50 ° C per almeno 1h. NOTA: incubazione degli embrioni durante la notte è accettabile. Prima di incubazione, sigillare i pozzetti con un foglio adesivo per evitare l&#39;evaporazione. </li></ol></li><li> ibridazione <ol><li> In un tubo separato aggiungere 0,1-0,3 ng / ml di sonda e 500 mg / ml di lievito tRNA in tampone di ibridazione, quindi vortice delicatamente per creare soluzione della sonda. tRNA lievito viene aggiunto alla soluzione della sonda di diminuire legame della sonda anti-senso non specifico. Riscaldare questa soluzione a 50 ° C, e il buffer aspirare ibridazione. </li><li> Mix embrioni pre-ibridizzati in 100 microlitri della soluzione della sonda, sigillare con un foglio adesivo, e ibridano a 50 ° C per 2 a 7 giorni a seconda della sonda (sonda foxq2 può essere incubato per 2 giorni). NOTA: i tempi di incubazione possono variare in base al largo della sonda. Alcuni geni espressi a livelli bassi richiedono fino a un&#39;incubazione di 7 giorni. piastre a 96 pozzetti possono essere collocati in una scatola di umidità come assicurazione contro l&#39;evaporazione se i ADHESScheda ive non riesce a sigillare correttamente. </li><li> Lavare 5 volte a 50 ° C con 180 ml di MOPS appena fatto il tampone di lavaggio per un totale di 3 ore. Poi, lavare 3 volte (15 min) con 180 ml di MOPS tampone di lavaggio a temperatura ambiente. Ricordarsi di mescolare gli embrioni nel tampone di lavaggio ogni volta pipettando delicatamente più volte. </li></ol></li><li> incubazione <ol><li> Aspirare il MOPS tampone di lavaggio e mescolare gli embrioni in 180 ml di tampone di bloccaggio composto da 10% di siero di pecora normale e 5 mg / ml di BSA in MOPS tampone di lavaggio e incubare per almeno 45 minuti a temperatura ambiente o 4 ° C durante la notte. </li><li> Rimuovere tampone di bloccaggio e poi mescolare gli embrioni in tampone di bloccaggio contenente una diluizione 1: 1.500 del alcalina anticorpo anti-digossigenina fosfatasi coniugato diluito in tampone di bloccaggio. Incubare per una notte a temperatura ambiente in un piatto sigillato per evitare l&#39;evaporazione. NOTA: Non lasciare anticorpo in più di una notte. </li><li> Lavare gli embrioni 6 volte (5 minuti o fino a quando gli embrioni drop) in MOPS tampone di lavaggio a temperatura ambiente. embrioni possonomemorizzare notte a 4 ° C. </li></ol></li><li> Nello sviluppo situ <ol><li> Lavare embrioni 3 volte (10 min) a temperatura ambiente con fresco tampone a pH 9.5 comprensivi di 0,1 M Tris pH 9,5, 100 mM NaCl, 50 mM MgCl 2, 1 mM Levamisolo, e 0,1% Tween-20. <ol><li> Per 20 mL pH 9.5 utilizzo del buffer 2 ml 1 M Tris pH 9.5, 400 ml 5M NaCl, 1 ml di 1 M MgCl 2, 20 microlitri Tween-20, 16,6 mL dH 2 O, e 0,0048 g Levamisolo. </li></ol></li><li> Incubare embrioni a 37 ° C in tampone colorazione riparo dalla luce. tampone colorazione consiste di 10% dimetilformammide, 4,5 microlitri / ml 4-nitro blu tetrazolio cloruro (NBT), e 3,5 microlitri / ml 5-Bromo-4-cloro-3-indolil-fosfato (BCIP) in appena fatta pH 9,5 tampone . <ol><li> Per 1 ml di tampone colorazione utilizzare il 100 microlitri dimetilformamide, 4,5 ml NBT, e 3,5 microlitri BCIP. </li></ol></li><li> Arrestare la reazione della fosfatasi alcalina lavando 3 a 5 volte in MOPS tampone di lavaggio. La connessione wi reazioneesimo la sonda foxq2 richiede in genere 30 minuti a 1 ora. Alcune sonde possono avere bisogno di incubazione durante la notte sia a RT o 4 ° C. </li><li> Mescolare embrioni in una soluzione di 70% di lavaggio MOPS e 30% glicerolo. Il glicerolo fornisce un indice di rifrazione necessaria per microscopia. Gli embrioni possono essere memorizzati in questa soluzione per diverse settimane. Sigillare le piastre con paraffina di plastica per evitare l&#39;evaporazione. </li></ol></li><li> Preparazione del vetrino e la cattura di immagini <ol><li> Preparare un vetrino organizzando tre piccole strisce di nastro biadesivo in un triangolo con piccoli spazi tra le strisce su un vetrino. </li><li> Trasferire gli embrioni nel lavaggio MOPS 70% e soluzione di glicerolo 30% al centro del triangolo e coprire con coprioggetto. </li><li> Sigillare il vetrino mediante l&#39;applicazione di uno strato di smalto intorno ai bordi del vetrino. </li><li> Catturare immagini utilizzando un microscopio ottico composto con un obiettivo 20X e una fotocamera collegata. </li></ol></li></ol>

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	<li>Erwin, D. H., Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+hierarchical+gene+regulatory+networks.">The evolution of hierarchical gene regulatory networks.</a> Nature reviews. Genetics. 10, 141-148 (2009).</li><li>Peter, I. S., Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+of+gene+regulatory+networks+controlling+body+plan+development.">Evolution of gene regulatory networks controlling body plan development.</a> Cell. 144, 970-985 (2011).</li><li>Borggrefe, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Notch+intracellular+domain+integrates+signals+from+Wnt,+Hedgehog,+TGFbeta/BMP+and+hypoxia+pathways.">The Notch intracellular domain integrates signals from Wnt, Hedgehog, TGFbeta/BMP and hypoxia pathways.</a> Biochimica et biophysica acta. 1863, 303-313 (2016).</li><li>Range, R. C., Angerer, R. C., Angerer, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+canonical+and+noncanonical+Wnt+signaling+pathways+patterns+the+neuroectoderm+along+the+anterior-posterior+axis+of+sea+urchin+embryos.">Integration of canonical and noncanonical Wnt signaling pathways patterns the neuroectoderm along the anterior-posterior axis of sea urchin embryos.</a> PLoS Biol. 11, e1001467 (2013).</li><li>Cleary, M. A., van Osch, G. J., Brama, P. A., Hellingman, C. A., Narcisi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF,+TGFbeta+and+Wnt+crosstalk:+embryonic+to+in+vitro+cartilage+development+from+mesenchymal+stem+cells.">FGF, TGFbeta and Wnt crosstalk: embryonic to in vitro cartilage development from mesenchymal stem cells.</a> Journal of tissue engineering and regenerative medicine. 9, 332-342 (2015).</li><li>Lapraz, F., 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=RTK+and+TGF-beta+signaling+pathways+genes+in+the+sea+urchin+genome.">RTK and TGF-beta signaling pathways genes in the sea urchin genome.</a> Dev Biol. 300, 132-152 (2006).</li><li>Pires-daSilva, A., Sommer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+signalling+pathways+in+animal+development.">The evolution of signalling pathways in animal development.</a> Nature reviews. Genetics. 4, 39-49 (2003).</li><li>Sethi, A. J., Wikramanayake, R. M., Angerer, R. C., Range, R. C., Angerer, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+signaling+crosstalk+regulates+endomesoderm+segregation+in+sea+urchin+embryos.">Sequential signaling crosstalk regulates endomesoderm segregation in sea urchin embryos.</a> Science. 335, 590-593 (2012).</li><li>Range, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+and+positioning+of+the+anterior+neuroectoderm+in+deuterostome+embryos.">Specification and positioning of the anterior neuroectoderm in deuterostome embryos.</a> Genesis. 52, 222-234 (2014).</li><li>Petersen, C. P., Reddien, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+and+the+polarity+of+the+primary+body+axis.">Wnt signaling and the polarity of the primary body axis.</a> Cell. 139, 1056-1068 (2009).</li><li>Lapraz, F., Haillot, E., 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=A+deuterostome+origin+of+the+Spemann+organiser+suggested+by+Nodal+and+ADMPs+functions+in+Echinoderms.">A deuterostome origin of the Spemann organiser suggested by Nodal and ADMPs functions in Echinoderms.</a> Nature communications. 6, 8434 (2015).</li><li>Kikuchi, A., Yamamoto, H., Sato, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+activation+mechanisms+of+Wnt+signaling+pathways.">Selective activation mechanisms of Wnt signaling pathways.</a> Trends in cell biology. 19, 119-129 (2009).</li><li>Hogan, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bone+morphogenetic+proteins:+multifunctional+regulators+of+vertebrate+development.">Bone morphogenetic proteins: multifunctional regulators of vertebrate development.</a> Genes Dev. 10, 1580-1594 (1996).</li><li>Houart, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+the+telencephalon+during+gastrulation+by+local+antagonism+of+Wnt+signaling.">Establishment of the telencephalon during gastrulation by local antagonism of Wnt signaling.</a> Neuron. 35, 255-265 (2002).</li><li>Bertrand, S., Escriva, H. <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:+amphioxus.">Evolutionary crossroads in developmental biology: amphioxus.</a> Development. 138, 4819-4830 (2011).</li><li>Rottinger, E., Lowe, C. J. <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:+hemichordates.">Evolutionary crossroads in developmental biology: hemichordates.</a> Development. 139, 2463-2475 (2012).</li><li>Genome Sequencing Sea Urchin, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+of+the+sea+urchin+Strongylocentrotus+purpuratus.">The genome of the sea urchin Strongylocentrotus purpuratus.</a> Science. 314, 941-952 (2006).</li><li>Ben-Tabou de-Leon, S., Su, Y. H., Lin, K. T., Li, E., Davidson, E. 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+control+in+the+sea+urchin+aboral+ectoderm:+spatial+initiation,+signaling+inputs,+and+cell+fate+lockdown.">Gene regulatory control in the sea urchin aboral ectoderm: spatial initiation, signaling inputs, and cell fate lockdown.</a> Dev Biol. 374, 245-254 (2013).</li><li>Saudemont, 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=Ancestral+regulatory+circuits+governing+ectoderm+patterning+downstream+of+Nodal+and+BMP2/4+revealed+by+gene+regulatory+network+analysis+in+an+echinoderm.">Ancestral regulatory circuits governing ectoderm patterning downstream of Nodal and BMP2/4 revealed by gene regulatory network analysis in an echinoderm.</a> PLoS Genet. 6, e1001259 (2010).</li><li>Cameron, R. A., Samanta, M., Yuan, A., He, D., Davidson, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SpBase:+the+sea+urchin+genome+database+and+web+site.">SpBase: the sea urchin genome database and web site.</a> Nucleic Acids Res. 37, D750-D754 (2009).</li><li>Stepicheva, N. A., Song, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+throughput+microinjections+of+sea+urchin+zygotes.">High throughput microinjections of sea urchin zygotes.</a> Journal of visualized experiments : JoVE. , e50841 (2014).</li><li>Cheers, M. S., Ettensohn, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+microinjection+of+fertilized+eggs.">Rapid microinjection of fertilized eggs.</a> Methods in cell biology. 74, 287-310 (2004).</li><li>Arenas-Mena, C., Cameron, A. R., Davidson, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+expression+of+Hox+cluster+genes+in+the+ontogeny+of+a+sea+urchin.">Spatial expression of Hox cluster genes in the ontogeny of a sea urchin.</a> Development. , 4631-4643 (2000).</li><li>Sethi, A. J., Angerer, R. C., Angerer, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multicolor+labeling+in+developmental+gene+regulatory+network+analysis.">Multicolor labeling in developmental gene regulatory network analysis.</a> Methods in molecular biology. , 249-262 (2014).</li><li>Wikramanayake, A. H., Huang, L., Klein, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=beta-Catenin+is+essential+for+patterning+the+maternally+specified+animal-vegetal+axis+in+the+sea+urchin+embryo.">beta-Catenin is essential for patterning the maternally specified animal-vegetal axis in the sea urchin embryo.</a> Proc Natl Acad Sci U S A. 95, 9343 (1998).</li><li>Yaguchi, S., Yaguchi, J., Angerer, R. C., Angerer, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Wnt-FoxQ2-nodal+pathway+links+primary+and+secondary+axis+specification+in+sea+urchin+embryos.">A Wnt-FoxQ2-nodal pathway links primary and secondary axis specification in sea urchin embryos.</a> Dev Cell. 14, 97-107 (2008).</li><li>Molina, M. D., de Croze, N., Haillot, E., 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:+master+and+commander+of+the+dorsal-ventral+and+left-right+axes+in+the+sea+urchin+embryo.">Nodal: master and commander of the dorsal-ventral and left-right axes in the sea urchin embryo.</a> Curr Opin Genet Dev. 23, 445-453 (2013).</li><li>Range, R. C., Glenn, T. D., Miranda, E., 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=LvNumb+works+synergistically+with+Notch+signaling+to+specify+non-skeletal+mesoderm+cells+in+the+sea+urchin+embryo.">LvNumb works synergistically with Notch signaling to specify non-skeletal mesoderm cells in the sea urchin embryo.</a> Development. 135, 2445-2454 (2008).</li><li>Range, R., 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=Cis-regulatory+analysis+of+nodal+and+maternal+control+of+dorsal-ventral+axis+formation+by+Univin,+a+TGF-beta+related+to+Vg1.">Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-beta related to Vg1.</a> Development. 134, 3649-3664 (2007).</li><li>Warner, J. F., Miranda, E. 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=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 metodologia qui presentata è un esempio che illustra il potere di usare embrioni con una minore complessità genomica e morfologica di vertebrati per capire le vie di segnalazione di trasduzione e GRN regolano meccanismi di sviluppo fondamentali .. Molti laboratori stanno utilizzando dosaggi simili durante lo sviluppo precoce riccio di mare a sezionare il coinvolti in percorsi di altri eventi di specifica destino della cellula di segnalazione (ad esempio Notch, Riccio, TGF-β, e la segnalazione FGF) <sup cl...

reti gene regolatore (GRN) e vie di trasduzione del segnale stabiliscono l&#39;espressione spaziale e temporale dei geni durante lo sviluppo embrionale, che vengono utilizzati per costruire il piano di adulti corpo animale. Cell-a-cella trasduzione del segnale sono componenti essenziali di queste reti di regolazione, fornendo i mezzi con cui le cellule comunicano. Queste interazioni cellulari stabilire e perfezionare l&#39;espressione di geni regolatori e differenziazione nei e tra i diversi territori durante l&#39;embriogenesi 1, 2. Le interazioni tra i modulatori secreti extracellulari (ligandi, antagonisti), recettori, e co-recettori controllano le attività delle vie di trasduzione del segnale. Un assortimento di molecole intracellulari transduces questi ingressi conseguente alterata espressione genica, divisione, e / o la forma di una cellula. Mentre molte delle molecole chiave utilizzati ai livelli extracellulari e intracellulari nelle principali vie sononoto, è una conoscenza incompleta dovuto in gran parte alla complessità delle vie di segnalazione individuali. Inoltre, diverse vie di segnalazione spesso interagiscono tra loro positivamente o negativamente al extracellulare, intracellulare e livelli trascrizionali 3, 4, 5, 6. È importante sottolineare che i componenti di base delle vie di trasduzione del segnale sono altamente conservati in tutte le specie metazoi, e, straordinariamente, la maggior parte delle vie di segnalazione spesso svolgono funzioni di sviluppo simili in molte specie quando si confrontano gli organismi da phyla strettamente correlati, in particolare 7, 8, 9, 10, 11. Lo studio di segnalazione durante lo sviluppo è un compito arduo in qualsiasi organismo, e cisono diverse sfide significative per studiare percorsi di segnalazione nella maggior parte dei modelli deuterostomi (vertebrati, cordati invertebrati, hemichordata, ed echinodermi): 1) Nei vertebrati ci sono un gran numero di possibili ligando e recettore / co-modulatore, molecole di trasduzione intracellulare, così come potenziali interazioni tra diverse vie di segnalazione a causa della complessità del genoma 12, 13, 14; 2) La morfologia complessa e movimenti morfogenetici nei vertebrati spesso rendono più difficile interpretare le interazioni funzionali e tra le vie di trasduzione del segnale; 3) Analisi in maggior parte delle specie modello invertebrato deuterostomi non echinoderma sono limitate da brevi finestre gravidity con l&#39;eccezione di alcune specie tunicate 15, 16. Ilriccio di mare embrione ha alcune delle limitazioni di cui sopra ed offre molte qualità uniche per l&#39;esecuzione di un&#39;analisi dettagliata delle vie di trasduzione del segnale in vivo. Questi sono i seguenti: 1) La relativa semplicità del genoma riccio riduce significativamente il numero di possibili ligando, recettore / co-recettore e molecola di trasduzione intracellulare interazioni 17; 2) I GRN che controllano la specifica e patterning degli strati germinali e le principali assi embrionali sono ben stabiliti in embrioni di riccio di mare, aiutando la comprensione del contesto normativo della cella / territorio, che riceve i segnali 18, 19; 3) Molti vie di trasduzione del segnale possono essere studiati tra gli stadi scissione e gastrula quando gli embrioni sono costituiti da un singolo strato dell&#39;epitelio la cui morfologia è più facile da analizzare; 4) Le molecole comportanod in vie di segnalazione nei ricci di mare sono facilmente manipolati; 5) Molti ricci di mare sono gravide per 10 a 11 mesi l&#39;anno (ad esempio Strongylocentrotus purpuratus e Lytechinus variegatus). Qui, vi presentiamo un metodo per caratterizzare in modo sistematico ed efficiente componenti delle vie di segnalazione che specificano e territori del modello negli embrioni di riccio di mare per illustrare i vantaggi che diversi sistemi modello invertebrati offrono nello studio dei meccanismi molecolari complessi.

<table><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 25 mg/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.

Nell&#39;embrione riccio di mare abbiamo dimostrato che 3 diverse di segnalazione Wnt rami (Wnt / β-catenina, Wnt / JNK, e Wnt / PKC) 4, 25 interagiscono per formare una rete di segnalazione Wnt che governa antero-posteriore (AP) patterning. Una delle più importanti conseguenze di questi eventi di segnalazione è che il ampiamente espresso anteriore neuroectoderma (ANE) GRN iniziale diventa limitato ad un piccolo territorio att...

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

Questo articolo descrive il video di una semplice metodologia vivo che può essere utilizzata per caratterizzare in modo sistematico ed efficiente componenti di vie di segnalazione complessi e reti di regolazione in molti embrioni invertebrati.

La forza della semplicità: Sea Urchin embrioni<em&gt; In Vivo</em&gt; Modelli di sviluppo per lo studio delle cellule cellula-Complex segnalazione di rete Interazioni

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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Cell Biology

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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 Views.  Mississippi State University.  Questo articolo descrive il video di una semplice metodologia vivo che può essere utilizzata per caratterizzare in modo sistematico ed efficiente componenti di vie di segnalazione complessi e reti di regolazione in molti embrioni invertebrati. 

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

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Microperla impianto nel zebrafish

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Ricerca

Biologia dello sviluppo

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Cellule staminali simil-<em&gt; Xenopus</em&gt; Embrionali espianti di studiare Caratteristiche precoce dello sviluppo neurale<em&gt; In Vitro</em&gt; E<em&gt; In Vivo</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

Embrione microiniezione e elettroporazione in chordate<em&gt; Intestinalis Ciona</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Immagini dal vivo della motilità cellulare e citoscheletro dei singoli neuroni e cellule della cresta neurale in embrioni di zebrafish

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Biologia

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Ibridazione dell'intero Monte In Situ negli embrioni di pesce zebra e nella formazione dei tubi, analisi dei prodotti iPSC-EC per studiare il ruolo dell'endoglin nello sviluppo vascolare

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

Microiniezione di embrioni Medaka per essere utilizzato come organismo modello genetico

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Proteomica della spettrometria di massa guidata da livrea cellulare nell'embrione in via di sviluppo (rana)

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

Chimica

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively na&#239;ve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise na&#239;ve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Zebrafish blastoderm explants are generated by isolating embryonic cells from endogenous signaling centers within the early embryo, producing relatively na&#239;ve cell clusters easily manipulated and cultured ex vivo. This article provides instructions for making such explants and demonstrates their utility by interrogating roles for Nodal signaling during gastrulation.

Generazione di espianti di blastoderma naïve da embrioni di zebrafish

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

La valutazione delle proprietà di segnalazione di ectodermica epiteli durante lo sviluppo craniofacciale

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

High Throughput microiniezioni di Sea Urchin zigoti

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

La forza della semplicità: Sea Urchin embrioni

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