وقد تم تمويل من قبل BBSRC هذا العمل منحة الهندسة الكهربائية والميكانيكية (BB / H010297 / 1)، حصة studentship BBSRC ريال، وstudentship MRC إلى 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.

جزء أساسي من هذا البروتوكول هو توليد بروابط نشطة بيولوجيا والتي تتم معالجتها عادة، يفرز، وقادرة على استثارة رد فعل في الخلية المستقبلة، على الرغم من وجود شاردة الفلورسنت المرفقة. فمن الأهمية بمكان لإثبات أن المنتج الجينات fluorescently الموسومة نشطة بيولوجيا باستخدام ال?...

أثناء التطور الجنيني المبكر، والخلايا التي ارتكبت تدريجيا لمتابعة الأنساب محددة من التمايز: وهذا يعني مجموعة من مكتملة النمو (أو المحفزة) الخلايا تصبح مقيدة تدريجيا إلى إقامة السكان من الخلايا الاصلية العزم على أن تؤدي إلى نوع خلية واحدة. إشارات الخلية خلية مركزية لتنظيم مواصفات النسب أثناء التطور الجنيني. سوف تكون هناك حاجة التلاعب في هذه الإشارات لتوجيه الخلايا الجذعية نحو مصائر خاصة لدعم العلاجات الطبية الجديدة. وأكد عدد صغير نسبيا من مسارات الإشارات خلال التنمية، بما في ذلك مسارات الاستجابة إلى الفصيلة TGF (nodals وأفضل الممارسات الإدارية) 1-2، عوامل النمو FGFs 3، 4 Wnts، والقنافذ 5. هذه البروتينات يفرز ربط مستقبلات موجودة على غشاء الخلية لتنشيط نقل الإشارة وبالتالي تغيير التعبير الجيني و / أو سلوك الخلية. تنظيم محكم من علامة الخليةalling أمر ضروري للمواصفات سلالة الخلايا والتطور الطبيعي. في حين أن الحديث المتبادل بين هذه الممرات مهم في تحديد مصير الخليوي، ويجند نفسه واحد يمكن أن تثير ردود متميزة في تركيزات مختلفة. وقد وصفت التدرجات محدثة التخلق منذ أكثر من 100 سنة بوصفها نظرية لشرح كيفية مختلف أنواع الخلايا يمكن أن تجنيها من حقل الخلايا 6. تشوير الجزيئات التي تنتجها مجموعة واحدة من الخلايا قد نشر على نطاق معين، وخفض التركيز في مسافة أكبر من المصدر. والخلايا المعرضة للإشارة لرد على تركيز المحلية في مركزها في مجال الخلايا، والخلايا في مواقع متميزة تستجيب بشكل مختلف لمستويات مختلفة من الإشارات. الدليل على وجود morphogens يأتي من الدراسات في وقت مبكر من ذبابة الفاكهة الجنين 7 و القرص الجناح 8، وكذلك الأطراف الفقارية 9 و 10 الأنبوب العصبي. وهناك حاجة إلى أساليب لفيvestigate كيف التدرجات محدثة التخلق يتم وضع وتحديد جزيئات أخرى مهمة في تنظيم هذه التدرجات. وقد استخدمت التجارب أنيقة باستخدام المناعية لتصور البروتينات الذاتية في الجسم الحي في سياق خلفيات وراثية مختلفة لتحقيق التدرجات محدثة التخلق 11-12. ومع ذلك، والأجسام المضادة جيدة والمسوخ محددة ليست متوفرة دائما، لذلك نحن هنا وصف البروتوكول باستخدام overexpression من بروابط الفلورسنت في القيطم، لتوفير بديل، طريقة بسيطة لتشريح كيفية منتجات الجين الخارجية يمكن أن تؤثر في توزيع بروابط عبر حقل من الخلايا. يوفر القيطم المورق نظام ممتاز لإجراء هذا النوع من التجارب مع تطور الأجنة من الخارج بحيث يمكن الوصول إليها في المراحل الأولى. حجمها كبير (1-1.5mm في القطر) يبسط microinjection والتلاعب الجراحية ومراحل الأريمة الخلايا هي سهلة لصورة لأنها لا تزال كبيرة نسبيا (حوالي 2081؛ م عبر). دراسات Overexpression في القيطم هي بسيطة للقيام: مرنا حقنها في الجنين في وقت مبكر يمكن أن تستهدف خلايا معينة، ويتم ترجمتها بكفاءة. تم إنشاء الموسومة fluorescently بنيات Wnt8a / Wnt11b-HA-EGFP باستخدام pCS2 Wnt8a-HA 13، pCS2 Wnt11b-HA 14 و EGFP. الببتيد HA مهم لتشمل، ليس فقط لتوفير علامة الجزيئية إضافية، ولكن أيضا لأنه يعتقد أن تتصرف كفاصل فصل البروتينات WNT وEGFP يسمح كل من المنتجات الجينات لتعمل. التركيبة المستخدمة لتصور Shh على استخدمت في السابق لتوليد الماوس المعدلة وراثيا تعبر عن Shh على-EGFP الانصهار البروتين 15؛ هذا وقدمت تفضلت اندي مكماهون. الأهم من ذلك، هو استنساخ العلامة GFP لجميع بنيات 3 &#39;إلى تسلسل إشارة بحيث يتم الاحتفاظ بها بعد المعالجة. ومن الضروري أيضا لضمان أن البروتين النهائي يتضمن تسلسل المطلوبة للتعديلات، مثل وadditioن من الدهون كما هو الحال بالنسبة لShh على وWNT ligands.The cDNAs تم subcloned في pCS2 + ناقلات التعبير الذي هو الأمثل لبرودوشن مرنا الاصطناعية. ويشمل المروج SP6 وإشارة تذييل بعديد الأدينيلات (http://sitemaker.umich.edu/dlturner.vectors). العمل الموصوف هنا يوفر بروتوكول بسيط للمقارنة بين إفراز ونشر الموسومة fluorescently WNT وShh على الموسومة بروابط. عن طريق حقن كميات محددة من مرنا الاصطناعية، وبروتوكول circumnavigates أي مشاكل المرتبطة التعبير متغير من نواقل مختلفة استخدام منشطات مختلفة. وقد تم مؤخرا تطبيق هذه الأساليب لتحقيق في الآثار المترتبة على سلفات endosulfatase heparan Sulf1 على Shh على-EGFP وWnt8a / إفراز Wnt11b-HA-EGFP ونشرها في القيطم 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

يصف هذا البروتوكول وسيلة لتصور بروابط توزيعها عبر حقل من الخلايا. سهولة التعبير عن البروتينات الخارجية، جنبا إلى جنب مع حجم كبير من الخلايا في الأجنة في وقت مبكر، وجعل القيطم المورق نموذجا مفيدا لتصور بروابط الموسومة GFP. يتم ترجمتها من mRNAs الاصطناعية بكفاءة بعد الحقن في مرحلة مبكرة أجنة القيطم، والحقن يمكن أن تستهدف خلية واحدة. عندما جنبا إلى جنب مع التتبع النسب مثل غشاء المربوطة طلب تقديم العروض، وحقن الخلايا (ونسله) التي تنتج البروتين overexpressed يمكن بسهولة أن يتبع. يصف هذا البروتوكول وسيلة لإنتاج الموسومة من fluorescently WNT وShh على بروابط من حقن مرنا. أساليب تنطوي على ميلCRO تشريح إإكسبلنتس الأدمة (القبعات الحيوان) وتحليل يجند نشر في عينات متعددة. باستخدام التصوير متحد البؤر، معلومات عن إفراز يجند ونشر أكثر من مجال الخلايا يمكن الحصول عليها. التحليلات الإحصائية للصور مبائر توفر البيانات الكمية على شكل تدرجات يجند. قد تكون هذه الطرق مفيدة للباحثين الذين يرغبون في اختبار آثار العوامل التي يمكن أن تنظم شكل تدرجات محدثة التخلق.

يقدم تحليلا متحد البؤر من إإكسبلنتس الغطاء الحيواني معربا عن البروتينات الموسومة fluorescently نظام فعال لتصور توزيع يجند تحت ظروف تجريبية مختلفة. في مثال واحد، وتوزيع GFP الموسومة يظهر Shh على (الشكل 1). في المرحلة 2-خلية، يتم حقن أجنة القيطم في كل من الخلايا الت?...

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

مخطوطة يوفر هنا مجموعة بسيطة من طرق لتحليل إفراز ونشر بروابط الموسومة fluorescently في القيطم. ويوفر هذا السياق لاختبار قدرة بروتينات أخرى لتعديل توزيع يجند والسماح التجارب التي قد تعطي نظرة ثاقبة آليات تنظيم التدرجات محدثة التخلق.

باستخدام تحليل متحد البؤر من<em&gt; القيطم المورق</em&gt; بالتحقيق المغيرون من WNT وShh على محدثة التخلق التدرجات

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 Views.  The Bateson Centre, University of Sheffield.  مخطوطة يوفر هنا مجموعة بسيطة من طرق لتحليل إفراز ونشر بروابط الموسومة fluorescently في القيطم. ويوفر هذا السياق لاختبار قدرة بروتينات أخرى لتعديل توزيع يجند والسماح التجارب التي قد تعطي نظرة ثاقبة آليات تنظيم التدرجات محدثة التخ...

Video: 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.

فهم توالد المبكر باستخدام المبسطة<em&gt; في الموقع</em&gt; بروتوكول التهجين في<em&gt; القيطم</em

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

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.

التلاعب و<em&gt; في المختبر</em&gt; نضوج<em&gt; القيطم المورق</em&gt; البويضات، تلاه سهم داخل الهيولى حقن الحيوانات المنوية، لدراسة التطور الجنيني

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.

الاستنساخ وظيفية باستخدام<em&gt; القيطم</em&gt; نظام التعبير البويضة

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.

Loss- ونهج كسب من وظيفة إلى التحقيق في خلية المبكر مصير محددات في سابق للانغراس أجنة الفئران

 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.

تقنية لاستهداف حقن مكروي إلى البلدان النامية<em&gt; القيطم</em&gt; الكلى

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.

دراسة إشارات Wnt خلال الزخرفة من إجراء الخطوط الجوية

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.

بناء نماذج العناصر المحدودة المعنية بالتحقيق في الزرد الفك الميكانيكا الحيوية

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.

التصور والتحليل الكمي من الأوعية الدموية الجنينية في<em&gt; زينوبوس تروبيكاليس</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.

الحقن المجهري للحمض النووي في براعم العيون في أجنة Xenopus laevis والتصوير من GFP التعبير عن الأشجار Axonal البصرية في سليمة، وحيا 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.

تصوير الكالسيوم الفلوري والتهجين في الموقع اللاحق لتوصيف السلائف العصبية في زينوبوس 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 ...