Name: multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos
Uploaded: 2017-08-09T15:00:00
Description: Watch this Scientific Journal Video about Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos at JoVE.com

Yazarlar Thomas Schilling nazikçe Tg (sox10:eGFP) Balık ve Mary Halloran nazikçe Tg(foxd3:GFP) balık hediye hediye için teşekkür ederiz.

The protocol described here was performed in accordance with the guidelines for the humane treatment of laboratory animals established by the University of Michigan Committee on the Use and Care of Animals (UCUCA).
1. Embryo Collection for Time-lapse Imaging
<ol>
	<li>Between 3 and 9 pm, set up male and female adult Tg(sox10:EGFP) or Tg(foxd3:GFP) transgenic zebrafish in a divided breeding tank for pairwise mating. 
	NOTE: The Tg(sox10:EGFP) and Tg(foxd3:G...

<ol>
	<li>Barembaum, M., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+steps+in+neural+crest+specification.">Early steps in neural crest specification.</a> Sem Cell Dev Biol. 16, 642-646 (2005).</li><li>Gage, P. J., Rhoades, W., Prucka, S. K., Hjalt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+maps+of+neural+crest+and+mesoderm+in+the+mammalian+eye.">Fate maps of neural crest and mesoderm in the mammalian eye.</a> Invest Ophthalmol Vis Sci. 46 (11), 4200-4208 (2005).</li><li>Minoux, M., Rijli, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+cranial+neural+crest+cell+migration+and+patterning+in+craniofacial+development.">Molecular mechanisms of cranial neural crest cell migration and patterning in craniofacial development.</a> Development. 137, 2605-2621 (2010).</li><li>Trainor, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+of+neural+crest+cell+formation+and+migration+in+mouse+embryos.">Specification of neural crest cell formation and migration in mouse embryos.</a> Sem Cell Dev Biol. 16, 683-693 (2005).</li><li>Johnston, M. C., Noden, D. M., Hazelton, R. D., Coulombre, J. L., Coulombre, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+avian+ocular+and+periocular+tissues.">Origins of avian ocular and periocular tissues.</a> Exp Eye Res. 29, 27-43 (1979).</li><li>Bohnsack, B. L., Kahana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thyroid+hormone+and+retinoic+acid+interact+to+regulate+zebrafish+craniofacial+neural+crest+development.">Thyroid hormone and retinoic acid interact to regulate zebrafish craniofacial neural crest development.</a> Dev Biol. 373, 300-309 (2013).</li><li>Chawla, B., Schley, E., Williams, A. L., Bohnsack, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+and+pitx2+regulate+early+neural+crest+survival+and+migration+in+craniofacial+and+ocular+development.">Retinoic acid and pitx2 regulate early neural crest survival and migration in craniofacial and ocular development.</a> Birth Defects Res B Dev Reprod Toxicol. , (2016).</li><li>Trainor, P. A., Tam, P. P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+paraxial+mesoderm+and+neural+crest+cells+of+the+mouse+embryo-codistribution+in+the+craniofacial+mesenchyme+but+distinct+segregation+in+branchial+arches.">Cranial paraxial mesoderm and neural crest cells of the mouse embryo-codistribution in the craniofacial mesenchyme but distinct segregation in branchial arches.</a> Development. 121 (8), 2569-2582 (1995).</li><li>Hong, C. S., Saint-Jeannet, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sox+proteins+and+neural+crest+development.">Sox proteins and neural crest development.</a> Sem Cell Dev Biol. 16, 694-703 (2005).</li><li>Steventon, B., Carona-Fontaine, C., Mayor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+network+during+neural+crest+induction:+from+cell+specification+to+cell+survival.">Genetic network during neural crest induction: from cell specification to cell survival.</a> Sem Cell Dev Biol. 16, 647-654 (2005).</li><li>Dressler, S., 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=Dental+and+craniofacial+anomalies+associated+wth+Axenfeld-Rieger+syndrome+with+PITX2+mutation.">Dental and craniofacial anomalies associated wth Axenfeld-Rieger syndrome with PITX2 mutation.</a> Case Report Med. , 621984 (2010).</li><li>Ozeki, H., Shirai, S., Ikeda, K., Ogura, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anomalies+associated+with+Axenfeld-Rieger+syndrome.">Anomalies associated with Axenfeld-Rieger syndrome.</a> Graefes Arch Clin Exp Ophthalmol. 237 (9), 730-734 (1999).</li><li>Strungaru, M. H., Dinu, I., Walter, 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=Genotype-phenotype+correlations+in+Axenfeld-Rieger+malformation+and+glaucoma+patients+with+FOXC1+and+PITX2+mutations.">Genotype-phenotype correlations in Axenfeld-Rieger malformation and glaucoma patients with FOXC1 and PITX2 mutations.</a> Invest Ophthalmol Vis Sci. 48, 228-237 (2007).</li><li>Tumer, Z., Bach-Holm, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Axenfeld-Rieger+syndrome+and+spectrum+of+Pitx2+and+Foxc1+mutations.">Axenfeld-Rieger syndrome and spectrum of Pitx2 and Foxc1 mutations.</a> Eur J Hum Genet. 17, 1527-1539 (2009).</li><li>Schoner, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrocephalus,+agenesis+of+the+corpus+callosum,+and+cleft+lip/palate+represent+frequent+associations+in+fetuses+with+Peters+plus+syndrome+and+B3GALTL+mutations.+Fetal+PPS+phenotypes,+expanded+by+Dandy+Walker+cyst+and+encephalocele.">Hydrocephalus, agenesis of the corpus callosum, and cleft lip/palate represent frequent associations in fetuses with Peters plus syndrome and B3GALTL mutations. Fetal PPS phenotypes, expanded by Dandy Walker cyst and encephalocele.</a> Prenat Diagn. 33 (1), 75-80 (2013).</li><li>Aliferis, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+nonsense+B3GALTL+mutation+confirms+Peters+plus+syndrome+in+a+patient+with+multiple+malformations+and+Peters+anomaly.">A novel nonsense B3GALTL mutation confirms Peters plus syndrome in a patient with multiple malformations and Peters anomaly.</a> Ophthalmic Genet. 31 (4), 205-208 (2010).</li><li>Lesnik Oberstein, ., S, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peters+Plus+syndrome+is+caused+by+mutations+in+B3GALTL,+a+putative+glycosyltransferase.">Peters Plus syndrome is caused by mutations in B3GALTL, a putative glycosyltransferase.</a> Am J Hum Genet. 79 (3), 562-566 (2006).</li><li>Brittijn, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+development+and+regeneration:+new+tools+for+biomedical+research.">Zebrafish development and regeneration: new tools for biomedical research.</a> Int J Dev Biol. 53, 835-850 (2009).</li><li>Bohnsack, B. L., Kasprick, D., Kish, P. E., Goldman, D., Kahana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+zebrafish+model+of+Axenfeld-Rieger+Syndrome+reveals+that+pitx2+regulation+by+retinoic+acid+is+essential+for+ocular+and+craniofacial+development.">A zebrafish model of Axenfeld-Rieger Syndrome reveals that pitx2 regulation by retinoic acid is essential for ocular and craniofacial development.</a> Invest Ophthalmol Vis Sci. 53 (1), 7-22 (2012).</li><li>Williams, A. L., Eason, J., Chawla, B., Bohnsack, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cyp1b1+regulates+ocular+fissure+closure+through+a+retinoic+acid-independent+pathway.">Cyp1b1 regulates ocular fissure closure through a retinoic acid-independent pathway.</a> Invest Ophthalmol Vis Sci. 58 (2), 1084-1097 (2017).</li><li>Curran, K., Raible, D. W., Lister, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foxd3+controls+melanophore+specification+in+the+zebrafish+neual+crest+by+regulation+of+Mitf.">Foxd3 controls melanophore specification in the zebrafish neual crest by regulation of Mitf.</a> Dev Biol. 332 (2), 408-417 (2009).</li><li>Dutton, K., Dutton, J. R., Pauliny, A., Kelsh, R. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+morpholino+phenocopy+of+the+colourless+mutant.">A morpholino phenocopy of the colourless mutant.</a> Genesis. 30 (3), 188-189 (2001).</li><li>Dutton, K. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+colourless+encodes+sox10+and+specifies+non-ectomesenchymal+neural+crest+fates.">Zebrafish colourless encodes sox10 and specifies non-ectomesenchymal neural crest fates.</a> Development. 128 (21), 4113-4125 (2001).</li><li>Kucenas, S., Takada, N., Park, H. C., Woodruff, E., Broadie, K., Appel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CNS-derived+glia+ensheath+peripheral+nerves+and+mediate+motor+root+development.">CNS-derived glia ensheath peripheral nerves and mediate motor root development.</a> Nat Neurosci. 11, 143-151 (2008).</li><li>Denk, W., Strickler, J., Webb, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+laser+scanning+fluorescence+microscopy.">Two-photon laser scanning fluorescence microscopy.</a> Science. 248 (4951), 73-76 (1990).</li><li>Helmchen, F., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+tissue+two-photon+microscopy.">Deep tissue two-photon microscopy.</a> Nat Methods. 2 (12), 932-940 (2005).</li><li>Denk, W., Delaney, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+and+functional+imaging+of+neurons+using+2-photon+laser+scanning+microscopy.">Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy.</a> J Neurosci Methods. 54 (2), 151-162 (1994).</li><li>Bohnsack, B. L., Gallina, D., Kahana, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phenothiourea+sensitizes+zebrafish+cranial+neural+crest+and+extraocular+muscle+developmnt+to+changes+in+retinoic+acid+and+insulin-like+growth+factor+signaling.">Phenothiourea sensitizes zebrafish cranial neural crest and extraocular muscle developmnt to changes in retinoic acid and insulin-like growth factor signaling.</a> PLoS ONE. 6, e22991 (2011).</li><li>Gfrerer, L., Dougherty, M., Laio, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+craniofacial+development+in+the+sox10:kaede+transgenic+zebrafish+line+using+time-lapse+confocal+microscopy.">Visualization of craniofacial development in the sox10:kaede transgenic zebrafish line using time-lapse confocal microscopy.</a> J Vis Exp. (79), e50525 (2013).</li><li>Lopez, A. L. R., Garcai, M. D., Dickinson, M. E., Larina, I. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+confocal+microscopy+of+the+developing+mouse+embryonic+yolk+sac+vasculature.">Live confocal microscopy of the developing mouse embryonic yolk sac vasculature.</a> Methods Mol Biol. 1214, 163-172 (2015).</li><li>McGurk, P. D., Lovely, C. B., Eberhart, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+craniofacial+morphogenesis+in+zebrafish+using+4D+confocal+microscopy.">Analyzing craniofacial morphogenesis in zebrafish using 4D confocal microscopy.</a> J Vis Exp. (83), e51190 (2014).</li><li>Nowotschin, S., Ferrer-Vaquer, A., Hadjantonakis, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+mouse+development+with+confocal+time-lapse+microscopy.">Imaging mouse development with confocal time-lapse microscopy.</a> Methods Enzymol. 476, 351-377 (2010).</li></ol>

Çoklu foton hızlandırılmış görüntüleme geçici ve göçmen hücre popülasyonlarının vivo izlenmesini sağlar. Bu güçlü teknik embriyonik süreçleri gerçek zamanlı eğitim için kullanılabilir ve bu da çalışmanın, bu yöntemi sonuçları nöral crest hücre göç ve kalkınma geçerli bilgisine gelişmiş. Önceki zaman hata görüntüleme çalışmaları genellikle mikroskobu tarama confocal lazer kullanmaktadır. 29 , 30 ,</s...

Konjenital göz hastalıkları çocukluk körlüğe neden olabilir ve kafatası nöral crest anormallikleri nedeniyle çoğu kez. Nöral crest hücreler Nöral tüp sizden ortaya çıkan ve vücut boyunca çok sayıda doku formu geçici kök hücreleri vardır. 1 , 2 , 3 , 4 , 5 nöral crest hücreleri, ön ve mesencephalon,'kemik ve kıkırdak midface ve frontal bölgelerde ve Iris, kornea, Trabeküler meshwork ve gözün ön segmentte sklera neden olmaktadır. 4 , 6 , 7 , 8 nöral crest hücrelerden faringeal arches rhombencephalon formu, çene ve kardiyak çıkış yolu. 1 , 3 , 4 , 9 , 10 çalışmalar nöral Crest için oküler katkıları ve Perioküler geliştirme, bu hücreler omurgalı gözü geliştirme önemini vurgulayan sermiştir. Nitekim, nöral crest hücre göç ve farklılaşma bozulma yol Fasiyal ve oküler anomaliler için Axenfeld-Rieger sendromu ve engin artı sendromu gibi. 11 , 12 , 13 , 14 , 15 , 16 , 17 böylece, geçiş, yayılması ve bu nöral crest hücrelerinin farklılaşması kapsamlı bir anlayış Konjenital göz hastalıklarının altında yatan karmaşıklığı görmemizi sağlar.
Zebra balığı türleri çalışmak için oküler gelişim, Zebra balığı göz memeli meslektaşlarına benzer yapılardır ve birçok genler örümceklerle Zebra balığı ve memeliler arasında korunmuş bir güçlü model organizmadır. 18 , 19 , 20 Ayrıca, Zebra balığı embriyo şeffaf ve yumurtlayan, göz geliştirme gerçek zamanlı görselleştirme kolaylaştırmak vardır.
Daha önce yayımlanmış iş genişletilmesi,6,7,20 nöral crest hücre göç desen çok foton floresan yeşil flüoresan protein (GFP) SRY (cinsiyet belirleme bölgesi Y) transkripsiyon kontrolü altında etiketlenmiş transgenik Zebra balığı satırlarındaki Imaging hızlandırılmış kullanarak tanımlanmıştır-kutusu 10 (sox10) veya Forkhead kutusunu D3 (foxd3) gen düzenleyici bölgeler. 21 , 22 , 23 , 24. çoklu foton Floresans hızlandırılmış görüntüleme lazer mikroskobu ile fluorophores edilen örneklerin yüksek çözünürlüklü, üç boyutlu görüntüleri yakalamak için uzun dalga boyu çok foton Floresans uyarma ile tarama gelişmiş optik teknikleri birleştiren güçlü bir teknik olduğunu. 25 , 26 , 27 çok foton lazer kullanımı artan doku penetrasyonu ve azalan fluorophore ağartma dahil olmak üzere standart confocal mikroskobu üzerinde belirgin avantajları vardır.
Bu yöntemi kullanarak, nöral crest hücre göç ve göçmen yolları zamanlama konusunda farklı iki ayrı nüfus oluşturulmaması, yani foxd3 pozitif nöral crest hücrelerde Perioküler Mezenşim ve gelişmekte olan göz ve sox10 pozitif nöral crest kraniyofasiyal Mezenşim hücreleri vardı. Bu yöntemle, Zebra balığı oküler ve Fasiyal nöral crest göç göç görselleştirmek için bir yaklaşım tanıttı geliştirme sırasında gerçek zamanlı olarak düzenlenmiş nöral crest geçiş gözlemlemek kolaylaştırır.
Bu iletişim kuralı Tg (sox10:EGFP) ve bir örnek olarak Tg (foxd3:GFP) transgenik Zebra balığı erken göz gelişimi sırasında hızlandırılmış videoları oluşturma hakkında bilgi sağlar. Bu iletişim kuralı daha da erken gelişme Zebra balığı nöral crest hücrelerden türetilmiş herhangi bir oküler ve Fasiyal yapısının yüksek çözünürlüklü, üç boyutlu, gerçek zamanlı görselleştirme için uygulanabilir. Ayrıca, bu yöntem diğer doku ve organların Zebra balığı ve diğer hayvan modelleri geliştirilmesi görüntüleme için daha da uygulanabilir.

<table border="0" cellpadding="0" cellspacing="0" width="919">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Breeding Tanks with Dividers</td>
			<td style="width:175px;">Aquaneering</td>
			<td style="width:104px;">ZHCT100</td>
			<td style="width:391px;">Crossing Tank Set (1.0-liter) Clear Polycarbonate with Lid and Insert</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">M205 FA Combi-Scope</td>
			<td style="width:175px;">Leica Microsystems CMS GmbH</td>
			<td style="width:104px;"></td>
			<td style="width:391px;">Stereofluorescence Microscope - FusionOptics and TripleBeam</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium Chloride</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">7760-5KG</td>
			<td style="width:391px;">Double PE sack. CAS No. 7647-14-5, EC Number 231-598-3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Potassium Chloride</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">1049380500</td>
			<td style="width:391px;">Potassium chloride 99.999 Suprapur. CAS No. 7447-40-7, EC Number 231-211-8.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Calcium Chloride Dihydrate</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:104px;">C79-500</td>
			<td style="width:391px;">Poly bottle; 500 g. CAS No. 10035-04-8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Magnesium Sulfate (Anhydrous)</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">MX0075-1</td>
			<td style="width:391px;">Poly bottle; 500 g. CAS No. 7487-88-9, EC Number 231-298-2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Methylene Blue</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">284-12</td>
			<td style="width:391px;">Glass bottle; 25 g. Powder, Certified Biological Stain</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Sodium Bicarbonate</td>
			<td style="width:175px;">Millipore (EMD)</td>
			<td style="width:104px;">SX0320-1</td>
			<td style="width:391px;">Poly bottle; 500 g. Powder, GR ACS. CAS No. 144-55-8, EC Number 205-633-8</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">N-Phenylthiourea</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">P7629-25G</td>
			<td style="width:391px;">&#62;98%. CAS Number 103-85-5, EC Number 203-151-2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">Dimethylsulfoxide</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:104px;">D8418-500ML</td>
			<td style="width:391px;">Molecular Biology grade. CAS Number 67-68-5, EC Number 200-664-3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Tricaine Methanesulfonate</td>
			<td style="width:175px;">Western Chemical Inc.</td>
			<td style="width:104px;">MS222</td>
			<td style="width:391px;">Tricaine-S</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Low-Melt Agarose</td>
			<td style="width:175px;">ISC Bioexpress</td>
			<td style="width:104px;">E-3112-25</td>
			<td style="width:391px;">GeneMate Sieve GQA Low Melt Agarose, 25 g</td>
		</tr>
		<tr height="21">
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			<td style="width:391px;">Circle cover glass. 25 mm diameter</td>
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multi-photon time lapse imaging to visualize development in real-time: visualization of migrating neural crest cells in zebrafish embryos

Konjenital göz ve kraniyofasiyal anomaliler nöral crest, vücut boyunca çok sayıda hücre tipleri ortaya çıkmasına göçmen kök hücreler geçici bir nüfusa aksamalar yansıtır. Biyoloji nöral Crest anlama okudu vivo içinde olabilir genetik olarak uysal modelleri ve gerçek zamanlı olarak yansıtan sınırlı olmuştur. Zebra balığı nöral crest gibi göçmen hücre popülasyonlarının çalışmak için özellikle önemli bir gelişim modelidir. Erken nöral crest farklılaşma düzenlenmesi ve işaretçileri nöral crest hücreler için büyük olasılıkla gösterir çok sayıda hayvan modellerinde gösterilmiştir sox10 ve foxd3 gibi nöral crest geçiş gelişmekte olan göz içine incelemek için lazer ile uzun dalga boyu çok foton Floresans uyarma mikroskobu tarama gelişmiş optik teknikleri bir arada gelişmekte olan gözlerinin içine transgenik Zebra balığı embriyolar, yani Tg (sox10:EGFP) ve Tg (foxd3:GFP), yüksek çözünürlüklü, üç boyutlu, gerçek zamanlı video yakalama için uygulanmıştır. Çoklu foton hızlandırılmış görüntüleme davranış ve erken göz gelişmesine katkıda bulunan iki nöral crest hücre popülasyonlarının göç desenleri ayırt için kullanıldı. Bu iletişim kuralı hızlandırılmış videoları, Zebra balığı nöral crest göç sırasında örnek olarak oluşturma hakkında bilgi sağlar ve daha fazla erken gelişme Zebra balığı ve diğer canlılar birçok yapıların görselleştirmek için uygulanabilir.

Çoklu foton Floresans hızlandırılmış görüntüleme kraniyofasiyal yapıları ve Tg (sox10:EGFP) ve (foxd3:GFP) Tg gözlerime ön segment Zebra balığı hatları ortaya çıkmasına kafatası nöral crest hücre geçiş desenler ortaya videoları bir dizi oluşturulur. Örnek olarak, sox10 -pozitif nöral crest hücreler arasında 12 ve 30 hpf Nöral tüp kenarından kraniyofasiyal bölgeye (Video 1, Resim 2...

Watch this Scientific Journal Video about Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos at JoVE.com

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Geliştirme gerçek zamanlı olarak görselleştirmek için çoklu foton zaman atlamalı görüntüleme: nöral Crest hücrelerde geçirme Zebra balığı embriyo görselleştirme

Lazer ile uzun dalga boyu çok foton Floresans uyarma mikroskobu tarama gelişmiş optik teknikleri bir arada Tg (sox10:EGFP) ve Tg (foxd3:GFP) Zebra balığı embriyo nöral crest göçün yüksek çözünürlüklü, üç boyutlu, gerçek zamanlı görüntüleme yakalamak için uygulanmıştır.

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

The overall goal of multi-photon time lapse imaging is to capture a high-resolution three dimensional real time images of migratory cell populations. This method can help answer key questions in developmental biology, such as mapping migration pathways of different cell populations. The main advantage of this technique over traditional confocal microscopy is that multi-photon lasers have deeper tissue penetration and decreased phototoxicity.This increases the usability in thicker tissues and allows for longer periods of image acquisition. To set up for embryo collection between 3:00 and 9:00 p.m. assemble breeding tanks and fill them with reverse osmosis, or RO water.Transfer up to three females and three males into opposing sides of the divided tank. The next morning, remove the divider shortly after the lights turn on in the vivarium. Allow undisturbed mating for 20 minutes or until sufficient numbers of embryos are produced.The embryos will be at the bottom of the breeding tank. When eggs are observed, lift the slotted inner tank out along with the fish, and quickly place them into a clean, solid outer tank filled with RO water. Use an egg collecting screen to collect eggs from the outer tank and transfer the eggs to Petri dishes containing 30 milliliters of 1X embryo medium.Incubate the collected eggs at 28.5 degrees Celsius. For transgenic embryos, at 24 hours post-fertilization or HPF, remove the dead eggs and assess the developmental stage of the embryos. Using forceps, remove the chorions from three to four embryos that express GFP.Under a dissecting fluorescence stereo microscope with a standard 460 to 490 nanometer band pass excitation filter, screen for GFP positive embryos. Transfer these embryos to a separate Petri dish containing 30 milliliters of fresh 1X embryo medium. After 20 HPF, place the screened and dechorionated GFP positive embryos in 0.003%PTU solution to inhibit pigmentation.Avoid initiating treatment earlier as it can have adverse effects on neural crest and neuroepithelial development. Add 0.4 grams of LowMelt Agarose powder to 20mL of 1x embryo medium. Heat the mixture for one to two minutes or until the solution is clear and all particles are dissolved.Aliquot approximately 1mL of the Agarose solution into fresh 1.5mL centrifuge tubes for storage. To set up the open bath chamber, place a small amount of high vacuum grease on the base of the open bath chamber. Place a circular glass cover slip onto the base and screw the top of the open bath chamber onto the base until it is tight.Pipette approximately 500-700 microliters of 2%Agarose solution into the open bath chamber until the base is approximately 3/4s filled. Wait 30 seconds to allow the Agarose to cool slightly without completely polymerizing and subsequently transfer a single embryo to the center of the base. Under a fluorescent stereo microscope, use a 10 microliter micro pipette tip to orient the embryo and position it near the bottom of the Agarose so that it doesn't float away when the embryo medium is added.A critical step is to make sure that the embryo is positioned correctly in the Agarose. In these experiments, we orient the embryo laterally but the embryo can also be positioned to focus on dorsal and ventral areas. Monitor and reposition the embryo until the Agarose has set.Then use a transfer pipette to entirely fill the assembled open chamber with time-lapse embryo medium. After placing the entire set up onto the stage of the microscope, according to the text protocol, use the five times objective to locate the embryo. Then manually raise the stage to the highest position and use the fine focus to position the embryo in the middle of the microscope range.Manually lower the stage and change the five time objective to the 25 time water immersion objective. Carefully raise the stage to bring the embryo back into focus. In the software, click on the XYTZ mode for obtaining multiple images at time or T intervals in the XY plane over a depth of Z.Use the epifluorescence or bright field view to find the depth of focus in the area of interest which will demarcate the z-stack.For these experiments, the lateral edge of the eye is the beginning of the z-stack. When focused here, in the software click the begin button. After focusing down to the mid-line of the embryo, which is the end of the z-stack, click the end button.A critical step is to make sure that the Z step encompasses the area of interest. In our case, we want to capture the width of the eye from the lateral to the medial edges. Click on the menu for adjusting the acquisition time and the imaging frequency.For adequate recovery of the fluor4 and survival of the embryo, allow for a ratio of at least one to three between z-stack acquisition when laser power is on and recovery time when laser power is off. With appropriate time for embryo recovery, set the time between z-stacks in the designated window. Then set the total length of time for the experiment in the appropriate window.Now, turn on the live image setting to make final adjustments to the laser settings. Adjust laser transmission, gain and offset slider bars within the software to optimize the fluorescence image. Also, adjust the orientation of the embryo as needed depending on the length of the experiment, anticipated growth of the embryo, et cetera.Make sure that the area of interest remains within the frame through the duration of the experiment. During time-lapse acquisition, turn off the epifluorescence light source, cover the stage with the laser safety box which is adequate for protection against background light, then press start. Access the open bath chamber through the sliding doors on the laser safety box to refill it with time lapse embryo medium every 8-12 hours.Following imaging acquisition, open the files in the image processing software, highlight the correct image series. In the software, choose the process menu, click on 3D deconvolution and apply to deconvolve each z-stack. Import individual tif files into video processing software, then select all tif files and drag them into the video editor.Adjust the length of each image within the video to 0.1 seconds. Finally, export the video as a mov or mp4 file. Seen here by multi-photon fluorescence imaging are the migration patterns of cranial neural crest cells that give rise to the cranial facial structures and anterior segment of the eye in transgenic sox10:EGFP and foxd3:GFP zebrafish lines.Sox 10 positive neural crest cells between 12 and 30 hpf migrate from the edge of the neural tube into the cranial facial region. The cells from the prosencephalon and mesencephalon migrate dorsally and ventrally to the developing eye to populate the periocular mesencine. As indicated here, the sox10 positive cells form the frontal nasal process.Neural crest cells from the rhombencephalon migrate ventrally and give rise to the pharyngeal arches. Time lapse imaging of foxd3 positive neural crest cells between 30 and 60 hpf show that these cells migrated between the optic cup and surface ectoderm and though the ocular fissure marked by the arrow and coalesced around the lens marked by the asterisk forming the iris stroma. Once mastered, this technique from start to finish can be done in four days if it is performed properly.When attempting this procedure, it is important to remember to choose embryos with a high amount of fluorescence. Setting up the software initially can be challenging but once the parameters are determined, additional changes are typically minimal. After watching this video, you should have a good understanding of how to use multi-photon time-lapse microscopy to image migrating cells in the embryo.

Research

JoVE Journal

Developmental Biology

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Analiz<em&gt; İn Vivo</em&gt; Hücre Göç Zebra balığı Embriyolar hücre nakli ve Time-lapse Görüntüleme kullanılarak

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

Gelişim Biyolojisi

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

diseksiyon mikroskobu kullanılarak Time-lapse Görüntüleme ile Zebra balığı Böbrek Kalkınma Analizi Optik Kesit için donatılmış

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Kinetic Measurement and Real Time Visualization of Somatic Reprogramming

Somatik yeniden programlanması Kinetik Ölçümü ve Gerçek Zamanlı Görselleştirme

Somatic reprogramming has enabled the conversion of adult cells to induced pluripotent stem cells (iPSC) from diverse genetic backgrounds and disease ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Kurbağa ve Zebra balığı Embriyolarında Hipoksinin İndüklenmesi

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Zebra balığı embriyo geliştirilmesinde bir Vitro tahlil hematopoetik kök ve yaratıcı Quantitate gelişimi (HSPCs) hücreleri

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Kalsiyum akını, gözenek oluşumu ve P2X7 reseptörleri yetişkin nöral progenitör hücreler tarafından fagositoz araştırmak için gerçek zamanlı canlı hücre Akış Sitometresi

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...

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

Tüm Zebrabalığı Embriyolarının Uzun Zaman Atlamalı Konfokal Mikroskobu için Katmanlı Montaj Yöntemi

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

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Brainbow ve Time-lapse Konfokal Görüntüleme kullanarak Yaşayan Zebrabalıklarında Gelişen Beyni Görselleştirme

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Isolation, Culture, and Adipogenic Induction of Neural Crest Original Adipose-Derived Stem Cells from Periaortic Adipose Tissue

Periaortik Yağ Dokusundan Nöral Adipoz-Türetilmiş Kök Hücrelerin İzolasyon, Kültür ve Adipojenik İndüksiyonu

An excessive amount of adipose tissue surrounding the blood vessels (perivascular adipose tissue, also known as PVAT) is associated with a high risk of ...