Gli autori ringraziano Adeline Dolly per le foto di espianto di fibronectina, Eric Theveneau per utili discussioni e il Fondo Animal dell&#39;Institut Curie. CM è un borsista postdottorato della Regione Ile de France (Domaine d&#39;interet Majeur Stelo Pole), Université Paris Sud (addetto Temporaire d&#39;Enseignement et de Recherche), e Agence Nationale de la Recherche. Questo lavoro è stato finanziato dalla Université Paris Sud (Attractivite 2011), il Centre National de la Recherche Scientifique (Programma d&#39;azione thématique et sur fonti di incentivi), Association pour la Recherche contre le Cancer di Grant SFI20101201882, Ligue contre le Cancer, e Agence Nationale de la Recherche (Agence Nationale de la Recherche programma Blanc).

Gli esperimenti sono conformi alla normativa nazionale ed europea sulla protezione degli animali utilizzati a fini scientifici e con i principi sanciti a livello internazionale di sostituzione, riduzione e perfezionamento. 1. Preparazione di piatti fibronectina rivestite 14 <ol><li> Pipettare 500 ml di 10 mg / ml grado fibronectina cultura diluito in 1x tampone fosfato (PBS) in un piatto di plastica standard di Petri (Ø 40 x 11 mm). Incubare a 37 ° C per 1 ora. Nota: se si utilizza piatti di vetro o vetrini per l&#39;imaging successiva, utilizzare un / soluzione fibronectina ml 100 mg. </li><li> Lavare tre volte con PBS 1x. </li><li> Sostituire PBS con 500 ml di 1x PBS/0.1% di sieroalbumina bovina (BSA). Incubare a 37 ° C per 30 min. </li><li> Lavare tre volte con PBS 1x. </li><li> Riempire con 1x PBS e conservare a 4 ° C fino all&#39;uso (di solito per 24-48 ore). </li></ol> 2. La dissezione degli espianti NC &lt;ol&gt; <li> Preparare 3/4 Normale Anfibio Medium (NAM) (110 mM NaCl, 2 mM KCl, 1 mM Ca (NO3) 2, 1 MgSO mm 4, 0.1 mM EDTA, 1 NaHCO mm 3, 0.2x PBS, 50 mg / ml gentamicina 10,15). Non tenere più di 1 settimana per la dissezione, conservare a 4 ° C. NAM Older può essere utilizzato per la preparazione di piatti dissezione agarosio rivestite (in basso). </li><li> Preparare 60 millimetri piastre Petri per la dissezione, rivestiti con 10 ml di 2% agarosio in 3/4 NAM. </li><li> Raccolte strumenti di dissezione: pipette Pasteur in plastica, un perno insetto migliore montato su supporto del perno (vedi tabella materiali per riferimento), un &quot;coltello sopracciglio&quot; (fatta con capelli sopracciglio inclusi in paraffina sulla punta di una pipetta Pasteur di vetro 16), due belle Pinza per dissezione, P1000 pipetta e suggerimenti, uno stereoscopio binocolo con ingrandimento 8-40X a 50, una sorgente di luce brillante con fibre ottiche e guide. Tutti gli strumenti di dissezione devono essere tenuti puliti, dopo ogni esperimento, sciacquare due volte con acqua distillata, una volta with 100% di etanolo e lasciare asciugare. Conservare lontano dalla polvere. </li><li> Compila il piatto dissezione con NAM fresco quasi al top. </li><li> Ottenere X. laevis embrioni secondo procedure standard 10 e li invecchiare fino a raggiungere neurula fase 16 (vedere Nieuwkoop e Faber tabella di sviluppo 17). </li><li> Posizionare fase 16 embrioni nel piatto dissezione con il pipetta Pasteur di plastica. </li><li> Eseguire tutti i passaggi successivi sotto l&#39;ambito binoculare. Rimuovere la membrana vitellina con due coppie di pinze. Fare attenzione a non danneggiare la parte dorsale degli embrioni. </li><li> Attendere fino a quando gli embrioni hanno raggiunto la fase 17 per iniziare la dissezione. Fase 17 è corretto poiché la cresta neurale è chiaramente distingue dal tubo neurale, in contrasto con le fasi precedenti. Dopo la tappa 17, NC inizia la migrazione e mescolanza con mesenchima circostante. </li><li> Scavare un piccolo foro nel agarosio con una pinza per fare una nicchia per gli embrioni per mantenereloro durante la dissezione. Posizionare un embrione dorsale verso l&#39;alto. Nota: modellare l&#39;argilla può essere utilizzato per mantenere gli embrioni invece scavando un buco nel agarosio. </li><li> Fare un&#39;incisione nella parte laterale della piega neurale anteriore con il coltello sopracciglio e il perno insetti. Fare attenzione a non andare troppo in profondità l&#39;embrione: lo scopo di tagliare 2-3 strati di cellule (mai raggiungere il profondo come il archenteron!). Durante l&#39;intero processo di dissezione, non permettono ai tessuti dissezionati di entrare in contatto con l&#39;interfaccia aria-liquido dal X. tessuti embrionali laevis sono lisate mediante tensione superficiale. </li><li> Fare una seconda incisione parallela nella parte più dorsale della piega neurale con gli stessi strumenti. </li><li> Se necessario, ruotare il piatto di circa 90 °. Effettuare una terza incisione perpendicolare con il coltello sopracciglio e il perno insetto, sul lato posteriore delle prime due incisioni. La NC è una massa densa di cellule sotto lo strato esterno pigmentato della ectoderma, lateraleal tubo neurale. </li><li> Utilizzare la punta del coltello sopracciglio per staccare lo strato pigmentato ectoderm dal sottostante, NC, a partire dal terzo bordo incisione. </li><li> Utilizzare il lato del coltello sopracciglio per staccare il tessuto NC partire dal terzo incisione. Il tessuto NC è piuttosto grigiastro, mentre il mesoderma cefalico sottostante è bianco. Separare con attenzione il NC dal mesoderma. </li><li> Staccare il tessuto NC anteriormente dalla vescicola ottica, e tagliare a questo livello per liberare l&#39;espianto. Come controllo per i primi esperimenti, controllando l&#39;espressione snail2 (un marcatore canonico NC) in alcuni espianti l&#39;ibridazione in situ è raccomandato. </li></ol> 3. Espianti placcatura su fibronectina <ol><li> Inserire nel piatto fibronectina rivestite con Modified Danilchick Medium (MDM, NaCl 53 mM, Na2CO 3 5 mm, K gluconato 4,5 mm, Na gluconato 32 mm, MgSO 4 1 mM, CaCl2 1 mM, BSA 0,1%, regolare il pH a 8,3 con 1 M Bicine <sup&gt; 18). </li><li> Trasferire gli espianti nel piatto fibronectina rivestite con una pipetta P1000. Non lasciare mai che gli espianti di toccare l&#39;interfaccia aria-liquido. Primo, pipettare po &#39;di liquido nella punta, poi gli espianti, poi alcuni di nuovo liquido. Non lasciare che l&#39;aria nella punta durante il trasferimento espianti nel piatto fibronectina rivestite. Una volta che la punta è all&#39;interno del liquido del piatto fibronectina rivestite, lasciare che gli espianti lentamente scendono per gravità, o spingere molto delicatamente. Evitare di espellere gli espianti troppo veloce. </li><li> Mettere 10-30 espianti nel piatto con il coltello sopracciglio. Evitare i lati del piatto, che non sono adeguate per l&#39;imaging successiva. </li><li> Cercate di non spostare la parabola fino a quando gli espianti hanno attaccato la fibronectina (ci vogliono circa 15-30 min). Se necessario, spostare il piatto con molta attenzione. </li><li> Porre la capsula in un incubatore refrigerato (tra 15-20 ° C, per la tabella delle temperature vedi 17). Quando posto a 15 ° C, le cellule are di solito la migrazione in modo efficiente e di scattering, dopo 6 ore di incubazione (questa tempistica può variare tra esperimenti). </li></ol> 4. L&#39;innesto espianti in embrioni Host <ol><li> Tagliare un bel vetrino di vetro in pezzi molto piccoli che utilizzano pinze grossolani. Le dimensioni dei pezzi dovrebbe essere di circa 1,5 mm 2. Immergere i pezzi in una capsula di Petri contenente tre quarti NAM o PBS usando le pinze. </li><li> Preparazione dell&#39;embrione ospitante: sezionare l&#39;ospite NC 3/4 NAM come descritto al punto 3), con particolare attenzione a non danneggiare l&#39;embrione di accoglienza durante la rimozione della membrana vitellina e sviscerare il NC. La tensione del tessuto tende ad aumentare la dimensione dell&#39;ablazione: lasciare l&#39;embrione ospite guar parzialmente mentre sezionare l&#39;embrione donatore. </li><li> In un altro piatto agarosio rivestite o in un&#39;altra parte dello stesso piatto, sezionare l&#39;espianto NC dall&#39;embrione donatore come descritto nel protocollo 3. Trasferire l&#39;innesto NC nella capsula contenente l&#39;host con cura, come descritto inProtocollo 3. </li><li> Porre immediatamente l&#39;espianto NC sulla zona ablazione host utilizzando una pinza. L&#39;espianto deve allegare parzialmente. Mettere un pezzo di vetro coprioggetto sul embrione innestato a mantenere l&#39;innesto in posizione. Scegliere un pezzo di vetro più grande di embrione per evitare di danneggiarlo. L&#39;embrione sarà un po &#39;appiattito. </li><li> Attendere almeno 15-20 minuti, quindi rimuovere delicatamente il vetrino. Lasciate che l&#39;embrione recuperare per altri 10 minuti e pipettare con cura in un piatto pulito riempito con 3/4 NAM, utilizzando la pipetta Pasteur di plastica. </li></ol>

<ol>
	<li>Sauka-Spengler, T., Bronner-Fraser, 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+gene+regulatory+network+orchestrates+neural+crest+formation.">A gene regulatory network orchestrates neural crest formation.</a> Nat. Rev. Mol. Cell Biol. 9 (7), 557-568 (2008).</li><li>Theveneau, E., Mayor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+delamination+and+migration:+from+epithelium-to-mesenchyme+transition+to+collective+cell+migration.">Neural crest delamination and migration: from epithelium-to-mesenchyme transition to collective cell migration.</a> Dev. Biol. 366 (1), 34-54 (2012).</li><li>Stuhlmiller, T. J., Garc&#237;a-Castro, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Current+perspectives+of+the+signaling+pathways+directing+neural+crest+induction.">Current perspectives of the signaling pathways directing neural crest induction.</a> Cell. Mol. Life Sci. 69 (22), 3715-3737 (2012).</li><li>Milet, C., Monsoro-Burq, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+induction+at+the+neural+plate+border+in+vertebrates.">Neural crest induction at the neural plate border in vertebrates.</a> Dev. Biol. 366 (1), 22-33 (2012).</li><li>Pegoraro, C., Monsoro-Burq, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+and+transcriptional+regulation+in+neural+crest+specification+and+migration:+lessons+from+Xenopus+embryos.">Signaling and transcriptional regulation in neural crest specification and migration: lessons from Xenopus embryos.</a> Dev. Biol. 2 (2), 247-259 (2013).</li><li>Etchevers, H. C., Amiel, J., Lyonnet, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Bases+of+Human+Neurocristopathies.">Molecular Bases of Human Neurocristopathies.</a> Neural Crest Induct. Different. 589, 213-234 (2005).</li><li>Nieuwkoop, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+organization+of+the+central+nervous+system+in+amphibians.+Part+II.+Differentiation+and+organization.">Activation and organization of the central nervous system in amphibians. Part II. Differentiation and organization.</a> J. Exp. Zool. 120 (1), 33-81 (1952).</li><li>Nieuwkoop, P. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+and+organization+of+the+central+nervous+system+in+amphibians+Part+I.+Induction+and+activation.">Activation and organization of the central nervous system in amphibians Part I. Induction and activation.</a> J. Exp. Zool. 120 (1), 1-31 (1952).</li><li>Sharpe, C. R., Fritz, A., De Robertis, E. M., Gurdon, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homeobox-containing+marker+of+posterior+neural+differentiation+shows+the+importance+of+predetermination+in+neural+induction.">A homeobox-containing marker of posterior neural differentiation shows the importance of predetermination in neural induction.</a> Cell. 50 (5), 749-758 (1987).</li><li>Sive, H. L., Grainger, R. M., Harland, R. 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> Early development of Xenopus laevis: a laboratory manual. , (2000).</li><li>Monsoro-Burq, A. -. H., Fletcher, R. B., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+induction+by+paraxial+mesoderm+in+Xenopus+embryos+requires+FGF+signals.">Neural crest induction by paraxial mesoderm in Xenopus embryos requires FGF signals.</a> Development. 130 (14), 3111-3124 (2003).</li><li>Milet, C., Maczkowiak, F., Roche, D. D., Monsoro-Burq, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pax3+and+Zic1+drive+induction+and+differentiation+of+multipotent,+migratory,+and+functional+neural+crest+in+Xenopus+embryos.">Pax3 and Zic1 drive induction and differentiation of multipotent, migratory, and functional neural crest in Xenopus embryos.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (14), 5528-5533 (2013).</li><li>Sadaghiani, B., Thi&#233;baud, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+development+in+the+Xenopus+laevis+embryo,+studied+by+interspecific+transplantation+and+scanning+electron+microscopy.">Neural crest development in the Xenopus laevis embryo, studied by interspecific transplantation and scanning electron microscopy.</a> Dev. Biol. 124 (1), 91-110 (1987).</li><li>Theveneau, 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=Collective+chemotaxis+requires+contact-dependent+cell+polarity.">Collective chemotaxis requires contact-dependent cell polarity.</a> Dev. Cell. 19 (1), 39-53 (2010).</li><li>Slack, J. M., Forman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+interaction+between+dorsal+and+ventral+regions+of+the+marginal+zone+in+early+amphibian+embryos.">An interaction between dorsal and ventral regions of the marginal zone in early amphibian embryos.</a> J. Embryol. Exp. Morphol. 56, 283-299 (1980).</li><li>Sive, H. L., Grainger, R. M., Harland, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+dissection+and+micromanipulation+tools.">Embryo dissection and micromanipulation tools.</a> CSH Protoc. , (2007).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+table+of+Xenopus+laevis+(Daudin):+a+systematical+and+chronological+survey+of+the+development+from+the+fertilized+egg+till+the+end+of+metamorphosis.">Normal table of Xenopus laevis (Daudin): a systematical and chronological survey of the development from the fertilized egg till the end of metamorphosis.</a> , (1994).</li><li>Theveneau, E., Mayor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beads+on+the+run:+beads+as+alternative+tools+for+chemotaxis+assays.">Beads on the run: beads as alternative tools for chemotaxis assays.</a> Methods Mol. Biol. 769, 449-460 (2011).</li><li>Bronner, M. E., LeDouarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evolution+of+the+neural+crest:+An+overview.">Development and evolution of the neural crest: An overview.</a> Dev. Biol. 366 (1), 2-9 (2012).</li><li>Alfandari, D., Cousin, H., Gaultier, A., Hoffstrom, B. G., DeSimone, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrin+&#945;5&#946;1+supports+the+migration+of+Xenopus+cranial+neural+crest+on+fibronectin.">Integrin &#945;5&#946;1 supports the migration of Xenopus cranial neural crest on fibronectin.</a> Dev. Biol. 260 (2), 449-464 (2003).</li><li>Fort, P., 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=Activity+of+the+RhoU/Wrch1+GTPase+is+critical+for+cranial+neural+crest+cell+migration.">Activity of the RhoU/Wrch1 GTPase is critical for cranial neural crest cell migration.</a> Dev. Biol. 350 (2), 451-463 (2011).</li><li>Matthews, H. K., 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=Directional+migration+of+neural+crest+cells+in+vivo+is+regulated+by+Syndecan-4/Rac1+and+non-canonical+Wnt+signaling/RhoA.">Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA.</a> Development. 135 (10), 1771-1780 (2008).</li><li>Kashef, J., K&#246;hler, A., Kuriyama, S., Alfandari, D., Mayor, R., Wedlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-11+regulates+protrusive+activity+in+Xenopus+cranial+neural+crest+cells+upstream+of+Trio+and+the+small+GTPases.">Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases.</a> Genes Dev. 23 (12), 1393-1398 (2009).</li><li>Hwang, Y. -. S., Luo, T., Xu, Y., Sargent, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myosin-X+is+required+for+cranial+neural+crest+cell+migration+in+Xenopus+laevis.">Myosin-X is required for cranial neural crest cell migration in Xenopus laevis.</a> Dev. Dyn. 238 (10), 2522-2529 (2009).</li><li>Lee, G., 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=Modelling+pathogenesis+and+treatment+of+familial+dysautonomia+using+patient-specific+iPSCs.">Modelling pathogenesis and treatment of familial dysautonomia using patient-specific iPSCs.</a> Nature. 461 (7262), 402-406 (2009).</li><li>Mica, Y., Lee, G., Chambers, S. M., Tomishima, M. J., Studer, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Neural+Crest+Induction,+Melanocyte+Specification,+and+Disease-Related+Pigmentation+Defects+in+hESCs+and+Patient-Specific+iPSCs..">Modeling Neural Crest Induction, Melanocyte Specification, and Disease-Related Pigmentation Defects in hESCs and Patient-Specific iPSCs..</a> Cell Reports. 3 (4), 1140-1152 (2013).</li><li>Shnitsar, I., Borchers, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PTK7+recruits+dsh+to+regulate+neural+crest+migration.">PTK7 recruits dsh to regulate neural crest migration.</a> Development. 135 (24), 4015-4024 (2008).</li><li>Matthews, H. K., 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=Directional+migration+of+neural+crest+cells+in+vivo+is+regulated+by+Syndecan-4/Rac1+and+non-canonical+Wnt+signaling/RhoA.">Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA.</a> Development. 135 (10), 1771-1780 (2008).</li><li>Matthews, H. K., Broders-Bondon, F., Thiery, J. P., Mayor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt11r+is+required+for+cranial+neural+crest+migration.">Wnt11r is required for cranial neural crest migration.</a> Dev. Dyn. 237 (11), 3404-3409 (2008).</li><li>Carmona-Fontaine, 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=Contact+inhibition+of+locomotion+in+vivo+controls+neural+crest+directional+migration.">Contact inhibition of locomotion in vivo controls neural crest directional migration.</a> Nature. 456 (7224), 957-961 (2008).</li><li>Guiral, E. C., Faas, L., Pownall, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+migration+requires+the+activity+of+the+extracellular+sulphatases+XtSulf1+and+XtSulf2.">Neural crest migration requires the activity of the extracellular sulphatases XtSulf1 and XtSulf2.</a> Dev. Biol. 341 (2), 375-388 (2010).</li><li>Cousin, H., Abbruzzese, G., Kerdavid, E., Gaultier, A., Alfandari, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translocation+of+the+Cytoplasmic+Domain+of+ADAM13+to+the+Nucleus+Is+Essential+for+Calpain8-a+Expression+and+Cranial+Neural+Crest+Cell+Migration.">Translocation of the Cytoplasmic Domain of ADAM13 to the Nucleus Is Essential for Calpain8-a Expression and Cranial Neural Crest Cell Migration.</a> Dev. Cell. 20 (2), 256-263 (2011).</li><li>Borchers, A., David, R., Wedlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenopus+cadherin-11+restrains+cranial+neural+crest+migration+and+influences+neural+crest+specification.">Xenopus cadherin-11 restrains cranial neural crest migration and influences neural crest specification.</a> Development. 128 (16), 3049-3060 (2001).</li><li>Borchers, A., Epperlein, H. -. H., Wedlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+system+to+study+migratory+behavior+of+cranial+neural+crest+cells+in+Xenopus.">An assay system to study migratory behavior of cranial neural crest cells in Xenopus.</a> Dev. Genes Evol. 210 (4), 217-222 (2000).</li><li>Kerney, R., Gross, J. B., Hanken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Runx2+is+essential+for+larval+hyobranchial+cartilage+formation+in+Xenopus+laevis.">Runx2 is essential for larval hyobranchial cartilage formation in Xenopus laevis.</a> Dev. Dyn. 236 (6), 1650-1662 (2007).</li><li>Yan, C. Y. I., Skourides, P., Chang, C., Samba Brivanlou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Xenopus+hnRNP+expressed+in+neural+and+neural+crest+tissues.">a Xenopus hnRNP expressed in neural and neural crest tissues.</a> Dev. Dyn. 238 (1), 204-209 (2009).</li><li>Acloque, H., Adams, M. S., Fishwick, K., Bronner-Fraser, M., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transitions:+the+importance+of+changing+cell+state+in+development+and+disease.">Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease.</a> J. Clin. Invest. 119 (6), 1438-1449 (2009).</li></ol>

Gli autori non hanno nulla da rivelare.

Questo protocollo descrive una tecnica facile da espiantare premigratory cranica NC in X. laevis embrioni. Gli embrioni utilizzati per tali esperimenti devono essere robusti e guarire bene. Eliminare ogni partita di embrioni sani. Inoltre, crescere embrioni a diverse temperature (da 12 a 18-20 ° C), al fine di asportare cresta neurale nella fase 17 e non più tardi. Dopo stadio 17, cresta neurale può essere miscelata con mesoderma cefalico e non può essere rimosso completamente. Tessuti Xenopus guar...

La cresta neurale (NC) è una popolazione di cellule embrionali transitoria che emerge dal tubo neurale a fine neurulazione negli embrioni dei vertebrati. Gli eventi di segnalazione e genetici che controllano specifiche NC iniziano già nel gastrulazione. La NC è specificato al confine tra la neurale e ectoderma neurale non da segnali dai tessuti dorsali circostanti. Alla fine del neurulazione, celle NC subiscono una transizione epitelio-to-mesenchima (EMT) e migrano ampiamente nell&#39;embrione seguendo percorsi stereotipati rispondendo al circostante spunti guida. Una volta che hanno raggiunto la loro destinazione finale, si differenziano in una vasta gamma di derivati, ad esempio, i neuroni, glia, ossa, cartilagine e cellule pigmentate 1-5. A causa del loro contributo a molti tipi di cellule e tessuti embrionali, difetti in ogni fase di sviluppo celle NC, dalla induzione alla differenziazione finale, può causare sindromi congenite denominate neurocristopathies 6. Emanipolazione Xperimental del CN ​​sviluppare in fasi diverse - specifica, EMT, migrazione e differenziamento - permetterà di migliorare la nostra comprensione di neurocristopathies e permettere la progettazione di potenziali strategie terapeutiche. L&#39;embrione laevis Xenopus è un modello di scelta per studiare lo sviluppo NC. Un gran numero di embrioni sono facili da ottenere, e la fecondazione esterna dà accesso alle prime fasi di sviluppo. Molti strumenti sono a disposizione per manipolare sperimentalmente X. laevis sviluppo embrionale. Gene guadagno-di-funzione e atterramento sono facili da eseguire da microinjecting singole cellule dei primi blastulas. Tessuti embrionali possono essere tagliati per riassociazione in vitro 7-11 o saggi di back-innesto 12,13. In questo protocollo, si descrive come sezionare fuori premigratory cranica NC in X. laevis neurulas fine, prima della migrazione. Tali espianti possono essere coltivate su fibronectina-coated piastre per studiare la migrazione e la differenziazione in vitro controllati in condizioni sperimentali. Espianti NC possono anche essere innestati in normali o manipolato gli embrioni di accoglienza per studiare la loro migrazione e differenziamento in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="601">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Fibronectin from bovine plasma</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F4759-1MG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Bovine Serum Albumine. Fraction V</td>
			<td style="width:71px;">Euromedex</td>
			<td style="width:119px;">04-100-811-C</td>
			<td>Lower quality grade may&#160; be suitable for this application</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Stainless Steel Insect Pins. Size 000</td>
			<td style="width:71px;">FST</td>
			<td align="right" style="width:119px;">26001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Dumont #5 forceps 0.05 mm x 0.02 mm</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">11252-20</td>
			<td></td>
		</tr>
	</tbody>
</table>

dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation

La cresta neurale (NC) è una popolazione cellulare dorsale tubo neurale transitoria che subisce una transizione epitelio-to-mesenchima (EMT) alla fine di neurulazione, migra verso estesamente vari organi, e si differenzia in molti tipi di derivati ​​(neuroni, glia, cartilagine e osso, le cellule pigmentate ed endocrine). In questo protocollo, si descrive come sezionare il premigratory cranica NC da Xenopus laevis embrioni, al fine di studiare lo sviluppo NC in vivo e in vitro. Il modello di rana offre molti vantaggi per studiare lo sviluppo precoce; lotti abbondanti sono disponibili, gli embrioni si sviluppano rapidamente, il guadagno in vivo e la perdita di strategie funzionali consentono la manipolazione dell&#39;espressione genica prima di NC dissezione del donatore e / o embrioni di accoglienza. Gli espianti NC possono essere piastrate su fibronectina e utilizzati per studi in vitro. Essi possono essere coltivate per diversi giorni in un mezzo definito privo di siero. Abbiamo anche descritto come innestare espianti NC indietroin embrioni di accoglienza per lo studio della migrazione NC e la differenziazione in vivo.

Quando placcato in fibronectina, gli espianti cresta neurale attaccano rapidamente (15-30 min) e l&#39;espianto si diffonde piatta entro 2 ore (Figura 1A). Dopo 3-6 cellule hr cominciano a disperdersi. A 24 ore a 15 ° C, molte cellule hanno iniziato a migrare dalla espianto (Figure 1B e 1C). Tuorlo rende le cellule molto luminoso in contrasto di fase (Figura 1B). Sporgenze cellulari (filopodes e lamellipodes) sono chiaramente visibili dopo falloidina c...

Watch this Scientific Journal Video about Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation at JoVE.com

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

Questo protocollo descrive come sezionare premigratory cresta neurale cranica (NC) da Xenopus laevis neurulas. Tali espianti possono essere placcati in fibronectina e coltivate in vitro, o innestate di nuovo in embrioni di accoglienza. Questa tecnica consente di studiare i meccanismi di NC epitelio a mesenchima transizione, migrazione e differenziazione.

Dissezione dei<em&gt; Xenopus laevis</em&gt; Cresta neurale per<em&gt; In vitro</em&gt; Espianto Cultura o<em&gt; In vivo</em&gt; Trapianto

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

dissection-xenopus-laevis-neural-crest-for-vitro-explant-culture-or

Research

JoVE Journal

Biology

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

13.1K Views.  Centre Universitaire.  Questo protocollo descrive come sezionare premigratory cresta neurale cranica (NC) da Xenopus laevis neurulas. Tali espianti possono essere placcati in fibronectina e coltivate in vitro, o innestate di nuovo in embrioni di accoglienza. Questa tecnica consente di studiare i meccanismi di NC epitelio a mesenchima transizione, migrazione e differenziazione. 

Video: Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

This video demonstrates the technique used for preparation of organizer and animal pole explants from Xenopus laevis embryos, including the use of the eyebrow knife - a specialized dissection tool made of one's eyebrow. The protocol for assembling an adhesion assay is also given, which probes for the presence of key adhesion molecules present on the surface organizer or animal pole cells that are critical for proper development.

Dissezione di organizzatore e Espianti Polo animali da embrioni di Xenopus laevis e montaggio di un saggio Adesione Cellulare

Ricerca

Biologia

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Plastic sections maintain true tissue morphology in thin sections of tissue that can be immunostained with fluorescent secondary antibodies, making this method more useful than paraffin-embedded or frozen sections for many types of tissue. The method for staining, plastic embedding, and sectioning is demonstrated in this video.

Incorporare plastica e Sezionamento embrioni di Xenopus laevis

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, collect and dejelly the eggs, and sort the eggs to remove any damaged eggs.

Ottenere uova da Xenopus laevis Femmine

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Preparazione e frazionamento di Xenopus laevis Egg Estratti

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microiniezione degli ovociti di Xenopus Laevis

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development of a larger region of the embryo. This makes defining its role in a particular tissue difficult to analyze. For example, noggin overexpression in Xenopus laevis embryos causes the expansion of the entire anterior region, including the eye1,2. From this result, it is not known if Noggin plays a direct role in eye determination or that by causing an expansion of neural tissue, Noggin indirectly affects eye formation. Having this complex phenotype makes studying its eye-specific role in cell fate determination difficult to analyze. We have developed an assay that overcomes this problem. Taking advantage of the pluripotent nature of the Xenopus laevis animal cap 3, we have developed an assay to test the ability of gene product(s), like noggin or the eye field transcription factors (EFTFs), to transform caps into particular tissue or cell types by transplanting this tissue onto the side of the embryo 4. While we have found either Noggin protein treatment or a collection of transcription factors can determine retinal cell fate in animal caps, this procedure could be used to identify gene product(s) involved in specifying other tissues as well.

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Animal caps overexpressing gene product(s) are transplanted to the flank of developing Xenopus laevis embryos in order to establish whether tissue is determined.

Determinazione del tessuto Utilizzando il trapianto Cap animali (ACT) Assay in Xenopus laevis</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later stages of embryonic development, and to define how enhancers and promoters regulate gene expression within the embryo. The protocol demonstrated here can be used to efficiently produce transgenic Xenopus laevis embryos. This transgenesis approach involves three parts: 1. Sperm nuclei are isolated from adult X. laevis testis by treatment with lysolecithin, which permeabilizes the sperm plasma membrane. 2. Egg extract is prepared by low speed centrifugation, addition of calcium to cause the extract to progress to interphase of the cell cycle, and a high-speed centrifugation to isolate interphase cytosol. 3. Nuclear transplantation: the nuclei and extract are combined with the linearized plasmid DNA to be introduced as the transgene and a small amount of restriction enzyme. During a short reaction, egg extract partially decondenses the sperm chromatin and the restriction enzyme generates chromosomal breaks that promote recombination of the transgene into the genome. The treated sperm nuclei are then transplanted into unfertilized eggs. Integration of the transgene usually occurs prior to the first embryonic cleavage such that the resulting embryos are not chimeric. These embryos can be analyzed without any need to breed to the next generation, allowing for efficient and rapid generation of transgenic embryos for analyses of promoter and gene function. Adult X. laevis resulting from this procedure also propagate the transgene through the germline and can be used to generate lines of transgenic animals for multiple purposes.

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

This video protocol demonstrates a method for generating transgenic Xenopus laevis by introduction of transgenes into sperm nuclei followed by nuclear transplantation into unfertilized eggs.

Produzione di transgenici Xenopus laevis Integrazione mediata da enzima di restrizione e trapianto nucleare

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Trapianti viso a<em&gt; Xenopus laevis</em&gt; Embrioni

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.