Ce travail a été financé par un BBSRC accorder aux MdPE (BB / H010297 / 1), un quota de stagiaire de BBSRC SAR, et une bourse d&#39;études de la MRC au format SWF.

<ol>
	<li>Shen, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+signaling:+developmental+roles+and+regulation.">Nodal signaling: developmental roles and regulation.</a> Development. 134 (6), 1023-1034 (2007).</li><li>Shimmi, O., Newfeld, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+extracellular+and+post-translational+regulation+of+TGF-beta+family+signalling+pathways.">New insights into extracellular and post-translational regulation of TGF-beta family signalling pathways.</a> J Biochem. 154 (1), 11-19 (2013).</li><li>Pownall, M. E., Isaacs, H. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> FGF Signalling in Vertebrate Development. 1, 1-75 (2010).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt/beta-catenin+signaling+in+development+and+disease.">Wnt/beta-catenin signaling in development and disease.</a> Cell. 127 (3), 469-480 (2006).</li><li>Ingham, P. W., McMahon, A. P. <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+in+animal+development:+paradigms+and+principles.">Hedgehog signaling in animal development: paradigms and principles.</a> Genes and Development. 15 (23), 3059-3087 (2001).</li><li>Wolpert, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+hundred+years+of+positional+information.">One hundred years of positional information.</a> Trends in Genetics. 12 (9), 359-3564 (1996).</li><li>Rushlow, C. A., Shvartsman, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+dynamics,+spatial+range,+and+transcriptional+interpretation+of+the+Dorsal+morphogen+gradient.">Temporal dynamics, spatial range, and transcriptional interpretation of the Dorsal morphogen gradient.</a> Current Opinion in Genetics and Development. 22 (6), 542-546 (2012).</li><li>Erickson, 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=Formation+and+maintenance+of+morphogen+gradients:+an+essential+role+for+the+endomembrane+system+in+Drosophila+melanogaster+wing+development.">Formation and maintenance of morphogen gradients: an essential role for the endomembrane system in Drosophila melanogaster wing development.</a> Fly (Austin). 5 (3), 266-271 (2011).</li><li>Towers, M., Wolpert, L., Tickle, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gradients+of+signalling+in+the+developing+limb.">Gradients of signalling in the developing limb.</a> Current Opinions in Cell Biology. 24 (2), 181-187 (2012).</li><li>Dessaud, E., McMahon, A. P., Briscoe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pattern+formation+in+the+vertebrate+neural+tube:+a+sonic+hedgehog+morphogen-regulated+transcriptional+network.">Pattern formation in the vertebrate neural tube: a sonic hedgehog morphogen-regulated transcriptional network.</a> Development. 135 (15), 2489-2503 (2008).</li><li>Briscoe, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeobox+gene+Nkx2.2+and+specification+of+neuronal+identity+by+graded+Sonic+hedgehog+signalling.">Homeobox gene Nkx2.2 and specification of neuronal identity by graded Sonic hedgehog signalling.</a> Nature. 398 (6728), 622-627 (1999).</li><li>Ribes, V., 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=Distinct+Sonic+Hedgehog+signaling+dynamics+specify+floor+plate+and+ventral+neuronal+progenitors+in+the+vertebrate+neural+tube.">Distinct Sonic Hedgehog signaling dynamics specify floor plate and ventral neuronal progenitors in the vertebrate neural tube.</a> Genes and Development. 24 (11), 1186-1200 (2010).</li><li>Freeman, S. D., Moore, W. M., Guiral, E. C., Holme, A., Turnbull, J. E., 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=Extracellular+regulation+of+developmental+cell+signaling+by+XtSulf1.">Extracellular regulation of developmental cell signaling by XtSulf1.</a> Developmental Biology. 320 (2), 436-445 (2008).</li><li>Tao, Q., 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=Maternal+wnt11+activates+the+canonical+wnt+signaling+pathway+required+for+axis+formation+in+Xenopus+embryos.">Maternal wnt11 activates the canonical wnt signaling pathway required for axis formation in Xenopus embryos.</a> Cell. 120 (6), 857-871 (2005).</li><li>Chamberlain, C. E., Jeong, J., Guo, C., Allen, B. L., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Notochord-derived+Shh+concentrates+in+close+association+with+the+apically+positioned+basal+body+in+neural+target+cells+and+forms+a+dynamic+gradient+during+neural+patterning.">Notochord-derived Shh concentrates in close association with the apically positioned basal body in neural target cells and forms a dynamic gradient during neural patterning.</a> Development. 135 (6), 1097-1106 (2008).</li><li>Ramsbottom, S. A., Maguire, R. J., Fellgett, S. W., 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=Sulf1+influences+the+Shh+morphogen+gradient+during+the+dorsal+ventral+patterning+of+the+neural+tube+in+Xenopus+tropicalis.">Sulf1 influences the Shh morphogen gradient during the dorsal ventral patterning of the neural tube in Xenopus tropicalis.</a> Developmental Biology. 391 (2), 207-218 (2014).</li><li>Fellgett, S. W., Maguire, R. J., 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=Sulf1+has+ligand+dependent+effects+on+canonical+and+non-canonical+WNT+signalling.">Sulf1 has ligand dependent effects on canonical and non-canonical WNT signalling.</a> Journal of Cell Science. 128 (7), 1408-1421 (2015).</li><li>Dytham, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Choosing and using statistics : A biologist's guide. , (2005).</li><li>Fay, M. P., Proschan, 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=Wilcoxon-Mann-Whitney+or+t-test?+On+assumptions+for+hypothesis+tests+and+multiple+interpretations+of+decision+rules.">Wilcoxon-Mann-Whitney or t-test? On assumptions for hypothesis tests and multiple interpretations of decision rules.</a> Statistical Surveys. 4, 1-39 (2010).</li><li>Christian, J. L., McMahon, J. A., McMahon, A. P., Moon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xwnt-8,+a+Xenopus+Wnt-1+/int-1-related+gene+responsive+to+mesoderm-inducing+growth+factors,+may+play+a+role+in+ventral+mesodermal+patterning+during+embryogenesis.">Xwnt-8, a Xenopus Wnt-1 /int-1-related gene responsive to mesoderm-inducing growth factors, may play a role in ventral mesodermal patterning during embryogenesis.</a> Development. 111 (4), 1045-1055 (1991).</li><li>Du, S. J., Purcell, S. M., Christian, J. L., McGrew, L. L., Moon, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+distinct+classes+and+functional+domains+of+Wnts+through+expression+of+wild-type+and+chimeric+proteins+in+Xenopus+embryos.">Identification of distinct classes and functional domains of Wnts through expression of wild-type and chimeric proteins in Xenopus embryos.</a> Molecular and Cellular Biology. 15 (5), 2625-2634 (1995).</li><li>Tada, M., Smith, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xwnt11+is+a+target+of+Xenopus+Brachyury:+regulation+of+gastrulation+movements+via+Dishevelled,+but+not+through+the+canonical+Wnt+pathway.">Xwnt11 is a target of Xenopus Brachyury: regulation of gastrulation movements via Dishevelled, but not through the canonical Wnt pathway.</a> Development. 127 (10), 2227-2238 (2000).</li><li>Williams, P. H., Hagemann, A., Gonzalez-Gaitan, M., Smith, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+long-range+movement+of+the+morphogen+Xnr2+in+the+Xenopus+embryo.">Visualizing long-range movement of the morphogen Xnr2 in the Xenopus embryo.</a> Current Biology. 14 (21), 1916-1923 (2004).</li><li>McDowell, N., Zorn, A. M., Crease, D. J., 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=Activin+has+direct+long-range+signalling+activity+and+can+form+a+concentration+gradient+by+diffusion.">Activin has direct long-range signalling activity and can form a concentration gradient by diffusion.</a> Current Biology. 7 (9), 671-681 (1997).</li><li>Chen, Y., Schier, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+Nodal+signal+Squint+functions+as+a+morphogen.">The zebrafish Nodal signal Squint functions as a morphogen.</a> Nature. 411 (6837), 607-610 (2001).</li><li>Miller, J. R., Rowning, B. A., Larabell, C. A., Yang-Snyder, J. A., Bates, R. L., Moon, R. T. <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+dorsal-ventral+axis+in+Xenopus+embryos+coincides+with+the+dorsal+enrichment+of+dishevelled+that+is+dependent+on+cortical+rotation.">Establishment of the dorsal-ventral axis in Xenopus embryos coincides with the dorsal enrichment of dishevelled that is dependent on cortical rotation.</a> Journal of Cell Biology. 146 (2), 427-437 (1999).</li><li>Schohl, A., Fagotto, F. B. e. t. a. -. c. a. t. e. n. i. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAPK+and+Smad+signaling+during+early+Xenopus+development.">MAPK and Smad signaling during early Xenopus development.</a> Development. 129 (1), 37-52 (2002).</li><li>Muller, 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=Differential+diffusivity+of+Nodal+and+Lefty+underlies+a+reaction-diffusion+patterning+system.">Differential diffusivity of Nodal and Lefty underlies a reaction-diffusion patterning system.</a> Science. 336, 721-724 (2012).</li><li>Yu, S. R., Burkhardt, M., Nowak, M., Ries, J., Petrasek, Z., Scholpp, S., Schwille, P., Brand, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fgf8+morphogen+gradient+forms+by+a+source-sink+mechanism+with+freely+diffusing+molecules.">Fgf8 morphogen gradient forms by a source-sink mechanism with freely diffusing molecules.</a> Nature. 461, 533-536 (2009).</li><li>Janda, C. Y., Waghray , D., Levin, A. M., Thomas, C., Garcia, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+of+Wnt+recognition+by+Frizzled.">Structural basis of Wnt recognition by Frizzled.</a> Science. 337, 59-64 (2012).</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. , (2010).</li></ol>

The authors have no competing financial interests.

Une partie essentielle de ce protocole est de générer des ligands biologiquement actifs qui sont normalement traitées, sécrétés, et capables de déclencher une réponse dans la cellule réceptrice, en dépit d&#39;un fragment fluorescent attachée. Il est essentiel d&#39;établir que le produit du gène étiquetées par fluorescence est biologiquement active avec un essai approprié. Pour Shh-GFP, la capacité d&#39;activer l&#39;expression de PTC1 16 a été confirmée. Wnt8a a une capacité r...

Au cours du développement embryonnaire précoce, les cellules sont progressivement engagés à suivre lignées spécifiques de différenciation: cela signifie un groupe de totipotentes (ou pluripotentes) cellules deviennent progressivement limité à établir des populations de cellules progénitrices déterminées pour donner naissance à un type de cellule. Signalisation cellule-cellule est au cœur de la régulation de la spécification de la lignée cours du développement embryonnaire. La manipulation de ces signaux sera nécessaire pour diriger les cellules souches vers destins particuliers pour soutenir nouveaux traitements médicaux. Un nombre relativement faible de voies de signalisation sont réitéré au cours du développement, y compris les voies répondant à la superfamille TGF (nodals et BMP) 1-2, FGF 3, Wnts 4, 5 et hérissons. Ces protéines sécrétées lient des récepteurs présents sur la membrane de la cellule pour activer la transduction du signal en modifiant ainsi l&#39;expression du gène et / ou le comportement des cellules. La réglementation stricte du signe de la celluleAlling est essentiel pour la spécification de la lignée cellulaire et le développement normal. Alors que la diaphonie entre ces voies est importante pour déterminer le destin des cellules, un seul ligand peut se solliciter des réponses distinctes à différentes concentrations. Gradients de morphogènes ont été décrits il ya plus de 100 ans comme une théorie pour expliquer comment les différents types de cellules peuvent dériver d&#39;un domaine de 6 cellules. Signalisation des molécules produites par un groupe de cellules peut se diffuser sur une certaine plage, la diminution de la concentration avec une plus grande distance de la source. Les cellules exposées au signal répondront à la concentration locale à leur position dans le domaine des cellules, avec des cellules à des positions distinctes répondant différemment à différents niveaux du signal. Preuve de l&#39;existence de morphogènes proviennent d&#39;études sur l&#39;embryon précoce Drosophila 7 et le disque de l&#39;aile 8, ainsi que la branche des vertébrés 9 et 10 tube neural. Les méthodes sont nécessaires pour envestigate comment gradients morphogène sont établis et d&#39;identifier d&#39;autres molécules importantes dans la régulation de ces gradients. Élégantes immunohistochimie en utilisant des expériences de visualiser in vivo des protéines endogènes dans le contexte de différents contextes génétiques ont été utilisées pour étudier les gradients de morphogènes 11-12. Toutefois, les bons anticorps et des mutants spécifiques ne sont pas toujours disponibles, de sorte que nous décrivons ici un protocole utilisant la surexpression de ligands fluorescents dans Xenopus, de fournir une alternative, méthode simple pour disséquer la façon dont les produits de gènes exogènes peuvent influencer la distribution de ligands à travers un champ de cellules. Xenopus laevis fournit un excellent système pour entreprendre ces types d&#39;expériences que leurs embryons se développent à l&#39;extérieur afin qu&#39;ils soient accessibles dès les premiers stades. Leur grande taille (1-1.5mm de diamètre) simplifie la micro-injection et la manipulation chirurgicale et par étapes, la blastula cellules sont faciles à l&#39;image car ils sont encore relativement grande (environ 2081; m de diamètre). Études de surexpression dans Xenopus sont simples à faire: ARNm injectés dans l&#39;embryon précoce peuvent être ciblés vers des cellules particulières et est efficacement traduits. Les constructions Wnt8a / Wnt11b-HA-eGFP fluorescence marqués ont été générés en utilisant pCS2 Wnt8a-HA 13, pCS2 Wnt11b-HA 14 et eGFP. Le peptide HA est important de comprendre, non seulement pour fournir une étiquette supplémentaire moléculaire, mais aussi parce que l&#39;on pense à agir comme une entretoise qui sépare les protéines Wnt et eGFP permettant à la fois des produits de gènes de fonctionner. Le produit d&#39;assemblage utilisé pour la visualisation de Shh a été précédemment utilisée pour générer une souris transgénique exprimant une protéine de fusion eGFP Shh-15; cela a été aimablement fourni par Andy McMahon. Fait important, l&#39;étiquette de GFP pour toutes les constructions est clonée en 3 &#39;de la séquence de signal de telle sorte qu&#39;il est retenu après le traitement. Il est également essentiel de veiller à ce que la protéine finale comprend des séquences nécessaires à la modification, par exemple supplén des lipides comme est le cas pour Shh et Wnt ligands ADNc ont été sous-cloné dans le vecteur d&#39;expression pCS2 + qui est optimisé pour la prodution de l&#39;ARNm synthétique; il comprend un promoteur SP6 et le signal de polyadénylation (http://sitemaker.umich.edu/dlturner.vectors). Le travail décrit ici fournit un protocole simple pour comparer la sécrétion et la diffusion de fluorescence étiqueté Wnt et Shh marqués ligands. En injectant des quantités définies de l&#39;ARNm synthétique, le protocole fait le tour des problèmes associés à l&#39;expression variable à partir de différents vecteurs utilisant différents promoteurs. Ces méthodes ont été récemment appliqué pour étudier les effets du sulfate d&#39;héparane endosulfatase Sulf1 sur Shh-eGFP et Wnt8a / Wnt11b-HA-eGFP la sécrétion et la diffusion chez Xenopus 16-17.

<table>
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Melford</td>
			<td>MB 1200</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium acetate</td>
			<td>Ambion</td>
			<td>From Megascript SP6 Kit AM 1330</td>
			<td></td>
		</tr>
		<tr>
			<td>Bicarbonate</td>
			<td>VWR International</td>
			<td>RC-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium nitrate</td>
			<td>Sigma</td>
			<td>C13961</td>
			<td></td>
		</tr>
		<tr>
			<td>Cap analog (m7G(5&#39;))</td>
			<td>Applied Biosystems</td>
			<td>AM 8050</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma</td>
			<td>C 2432</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;L-Cysteine hydrochloride monohydrate</td>
			<td>Sigma</td>
			<td>C7880</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTPs</td>
			<td>Invitrogen</td>
			<td>18427-013</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma</td>
			<td>O3690</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>VWR International</td>
			<td>20821.33</td>
			<td></td>
		</tr>
		<tr>
			<td>Ficoll 400</td>
			<td>Sigma</td>
			<td>F 4375</td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji image J software</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Free download http://fiji.sc/Fiji</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Melford</td>
			<td>G 0124</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Fisher Scientific</td>
			<td>A/0400/PB17</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass cover slips, No.1.5&#160;</td>
			<td>Scientific Laboratory Supplies</td>
			<td>22X22-SGJ3015. 22X50-SGJ3030&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass needle puller</td>
			<td>Narishige</td>
			<td>Narishige PC -10</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass pull needles</td>
			<td>Drummond Scientific</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>Human chronic gonadotropin (HCG)</td>
			<td>Intervet</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>P/7500/PB17</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium chloride (LiCl)</td>
			<td>Sigma</td>
			<td>L-7026</td>
			<td></td>
		</tr>
		<tr>
			<td>LSM710 and Zen software (2008-2010)</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab software</td>
			<td>Mathworks</td>
			<td></td>
			<td>http://uk.mathworks.com/</td>
		</tr>
		<tr>
			<td>Molecular grade water&#160;</td>
			<td>Fisher Scientific</td>
			<td>BP 2819-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail varnish&#160;</td>
			<td>Boots</td>
			<td>Bar code 3600530 373048</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectrophotometer</td>
			<td>Lab.tech International</td>
			<td>ND-1000 / ND8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish (55mm)</td>
			<td>VWR International</td>
			<td>391-0865</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol-chloroform</td>
			<td>Sigma</td>
			<td>P3803</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoshop software</td>
			<td>Adobe</td>
			<td>N/A</td>
			<td>http://www.photoshop.com/products</td>
		</tr>
		<tr>
			<td>High fidelity DNA polymerase and buffers&#160;</td>
			<td>Biolabs</td>
			<td>M0530S&#160; Buffer - M0531S</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Fisher Scientific</td>
			<td>P/4280/53</td>
			<td></td>
		</tr>
		<tr>
			<td>PVC insulation tape</td>
			<td>Onecall</td>
			<td>SH5006MPK</td>
			<td></td>
		</tr>
		<tr>
			<td>Gel extraction kit&#160;</td>
			<td>Qiagen</td>
			<td>S28704</td>
			<td></td>
		</tr>
		<tr>
			<td>Restriction enzymes buffers</td>
			<td>Roche</td>
			<td colspan="2">SuRE/CUT Buffer Set 11082 035 001</td>
		</tr>
		<tr>
			<td>RNAse-free DNAse</td>
			<td>Promega</td>
			<td>ME10A</td>
			<td></td>
		</tr>
		<tr>
			<td>Steel back single edge blades</td>
			<td>Personna</td>
			<td>66-0403-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Fisher Scientific</td>
			<td>27810.364</td>
			<td></td>
		</tr>
		<tr>
			<td>SP6 transcription kit</td>
			<td>Ambion</td>
			<td>AM1330</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides</td>
			<td>Thermo Fisher</td>
			<td>SHE 2505</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Invitrogen</td>
			<td>15504-020</td>
			<td></td>
		</tr>
		<tr>
			<td>Tungsten needles</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>homemade</td>
		</tr>
		<tr>
			<td>Zen lite software</td>
			<td>Carl Zeiss</td>
			<td>N/A</td>
			<td>Free download&#160; http://www.zeiss.co.uk/microscopy/en_gb/downloads/zen.html</td>
		</tr>
	</tbody>
</table>

using confocal analysis of xenopus laevis to investigate modulators of wnt and shh morphogen gradients

Ce protocole décrit un procédé pour visualiser des ligands distribués à travers un champ de cellules. La facilité de l&#39;expression de protéines exogènes, ainsi que la taille de leurs cellules dans des embryons précoces, faire Xenopus laevis un modèle utile pour visualiser des ligands GFP étiqueté. ARNm synthétiques sont efficacement convertis après l&#39;injection dans des embryons de xénope à un stade précoce, et les injections peuvent être ciblés pour une seule cellule. Lorsqu&#39;il est combiné avec un traceur de lignage comme membrane attaché DP, la cellule injectée (et ses descendants) qui sont la production de la protéine surexprimée peuvent facilement être suivies. Ce protocole décrit un procédé pour la production de fluorescence étiquetée Wnt et Shh ligands injecté à partir de l&#39;ARNm. Les procédés impliquent la micro dissection des explants ectodermiques (caps animales) et l&#39;analyse de la diffusion de ligand dans plusieurs échantillons. En utilisant l&#39;imagerie confocale, informations sur la sécrétion ligand et la diffusion sur un champ de cellules peut être obtenue. Les analyses statistiques des images confocales fournissent des données quantitatives sur la forme de gradients de ligands. Ces méthodes peuvent être utiles aux chercheurs qui veulent tester les effets des facteurs qui peuvent réglementer la forme de gradients de morphogènes.

Analyse confocale des explants de plafonnement des animaux exprimant des protéines par fluorescence taggés fournit un système efficace pour la visualisation de la distribution de ligand dans différentes conditions expérimentales. Dans un exemple, la distribution de la GFP étiqueté Shh est représenté (figure 1). Au stade 2 cellules, les embryons de Xenopus sont injectés dans les cellules avec soit avec un ARNm de commande ou avec l&#39;ARNm codant pour Sulf1, une enzyme qui modifie l&#...

Watch this Scientific Journal Video about Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients at JoVE.com

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Le manuscrit ici fournit un jeu simple de méthodes pour analyser la sécrétion et la diffusion de ligands marqués par fluorescence dans Xenopus. Ceci permet d&#39;obtenir un contexte pour tester la capacité des autres protéines de modifier la distribution de ligand et en permettant des expériences qui peuvent donner un aperçu des mécanismes de régulation des gradients de morphogènes.

Utilisation de l&#39;analyse confocale<em&gt; Xenopus laevis</em&gt; Chargé d&#39;enquêter sur les modulateurs de Wnt et Shh morphogène Dégradés

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

using-confocal-analysis-xenopus-laevis-to-investigate-modulators-wnt

Research

JoVE Journal

Developmental Biology

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

11.0K vues.  The Bateson Centre, University of Sheffield.  Le manuscrit ici fournit un jeu simple de méthodes pour analyser la sécrétion et la diffusion de ligands marqués par fluorescence dans Xenopus. Ceci permet d'obtenir un contexte pour tester la capacité des autres protéines de modifier la distribution de ligand et en permettant des expériences qui peuvent donner un aperçu des mécanismes de régulation des gradients de morphogènes. 

Vidéo: Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Comprendre organogenèse précoce aide d&#39;un simplifié<em&gt; In Situ</em&gt; Protocole hybridation in<em&gt; Xenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Recherche

Biologie du développement

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Manipulation et<em&gt; In Vitro</em&gt; Maturation du<em&gt; Xenopus laevis</em&gt; ovocytes, Suivi par injection intracytoplasmique de spermatozoïdes, pour étudier le développement embryonnaire

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Clonage fonctionnelle en utilisant un<em&gt; Xenopus</em&gt; Système d&#39;expression de l&#39;ovocyte

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

Déficitaires et approche Gain de fonction d&#39;enquêter sur les cellules précoce destin déterminants dans les embryons de souris préimplantatoire

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Technique à la cible microinjection du Developing<em&gt; Xenopus</em&gt; Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Studying Wnt Signaling During Patterning of Conducting Airways

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

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

Étude Wnt Signaling Pendant Patterning de Conduite Airways

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

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

Construire des modèles éléments finis pour enquêter sur Zebrafish Jaw Biomécanique

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Visualisation et analyse quantitative de l&#39;angiogenèse embryonnaire dans<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Microinjection d'ADN dans eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Imagerie de calcium fluorescent et hybridation in situ subséquente pour la caractérisation neuronale de précurseur dans Les laevis de Xenopus

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...