Name: visualization of chondrocyte intercalation and directional proliferation via zebrabow clonal cell analysis in the embryonic meckel&#8217;s cartilage
Uploaded: 2015-10-21T14:00:00
Description: Watch this Scientific Journal Video about Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckelâ€™s Cartilage at JoVE.

Forfatterne takker Alex Schier for venligt deler Zebrabow-M transgene linje, Geoffrey Burns for pDEST vektor og Renee Ethier-Daigle for fremragende pleje af fisken facilitet og linjer. FINANSIERING: Vi er taknemmelige for generøse finansiering støtte fra NIDCR RO3DE024490 og Shriners Hospitals for Children (ECL) og ph.d.-uddannelsesstipendier fra Shriners Hospitals for Children (LR og YK).

Massachusetts General Hospital Institutional Animal Care og brug Udvalg (IACUC) godkendte alle procedurer i henhold til protokol nummer # 2010N000106. Dette er i overensstemmelse med Foreningen for Vurdering og akkreditering af Laboratory Animal Care International (AAALAC) retningslinjer. 1. Reagenser og materialer Fremstilling <ol><li> Forbered 1 L 50X E3 embryo medium (se tabel 1) og forberede 1 L 1X E3 embryo medium (se tabel 2). </li><li> Forbered E3 e...

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	<li>Dougherty, M., 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+requirements+for+wnt9a+and+irf6+in+extension+and+integration+mechanisms+during+zebrafish+palate+morphogenesis.">Distinct requirements for wnt9a and irf6 in extension and integration mechanisms during zebrafish palate morphogenesis.</a> Development. 140, 76-81 (2013).</li><li>Dougherty, M., 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=Embryonic+fate+map+of+first+pharyngeal+arch+structures+in+the+sox10:+kaede+zebrafish+transgenic+model.">Embryonic fate map of first pharyngeal arch structures in the sox10: kaede zebrafish transgenic model.</a> The Journal of craniofacial surgery. 23, 1333-1337 (2012).</li><li>Eberhart, J. K., Swartz, M. E., Crump, J. G., Kimmel, C. 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Birth defects research.</a> Part C, Embryo today : reviews. 93, 67-114 (2011).</li><li>Mossey, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dental+education+and+CPD:+are+you+being+served.">Dental education and CPD: are you being served.</a> British dental journal. Suppl, 3-4 (2002).</li><li>Pan, Y. 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=Zebrabow:+multispectral+cell+labeling+for+cell+tracing+and+lineage+analysis+in+zebrafish.">Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish.</a> Development. 140, 2835-2846 (2013).</li><li>Vieira, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Journal of dental research. 87, 119-125 (2008).</li><li>Le Pabic, P., Ng, C., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fat-Dachsous+Signaling+Coordinates+Cartilage+Differentiation+and+Polarity+during+Craniofacial+Development.">Fat-Dachsous Signaling Coordinates Cartilage Differentiation and Polarity during Craniofacial Development.</a> PLoS genetics. 10, e1004726 (2014).</li><li>Ricks, J. E., Ryder, V. M., Bridgewater, L. C., Schaalje, B., Seegmiller, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+mandibular+development+precedes+the+time+of+palate+closure+in+mice+homozygous+for+disproportionate+micromelia:+an+oral+clefting+model+supporting+the+Pierre-Robin+sequence.">Altered mandibular development precedes the time of palate closure in mice homozygous for disproportionate micromelia: an oral clefting model supporting the Pierre-Robin sequence.</a> Teratology. 65, 116-120 (2002).</li><li>Javidan, Y., Schilling, T. 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Alcian Blue og photoconvertible transgene linier som beskrevet ovenfor supplerer hinanden for at definere den indviklede proces af brusk og knogle udvikling. Imidlertid har levende cellulære migration og organisation under organogenesen længe været en hypotese og indirekte demonstreret, men aldrig visualiseret. Zebrabow-M transgene linje kombineret med en brusk- specifikke Cre tillader samtidig levende observation af alle disse forskellige begivenheder involveret i knogle- og bruskdannelse. Denne teknik tillader stud...

Craniofacial udvikling kræver komplekse molekylære, cellulære og væv interaktioner til at køre celleproliferation, migration og differentiering 1,2, 3. Dette stramt reguleret og kompleks proces er underlagt genetiske og miljømæssige forstyrrelser, således at kraniofaciale misdannelser er blandt de mest almindelige medfødte misdannelser 1-9. Mens kirurgiske indgreb er fortsat grundpillen i behandling for kraniofaciale anomalier, forstå udviklingen grundlag er afgørende for at innovere fremtidige behandlinger. Derfor studere morfogenese og mekanismerne i konvergensprogrammet og udvidelse og celle integration giver nye indsigter i dannelsen af den kraniofacial skelet 1. Kraniel neurale våbenskjold migrere og befolke den første svælg bue, så danne parrede mandiblens processer, der strækker sig til at danne Meckel s brusk, som en forløber underkæben. Morfogenese of Meckel brusk kræver chondrocyt organisation via retningsbestemt proliferation, cellepolarisering og differentiering 1,10. Men forviklinger af chondrocytter organisation i vækst og udvidelse af Meckel brusk stadig uklar. Forståelse dynamiske celle adfærd er afgørende for forståelsen medfødte misdannelser, der påvirker mandibulær størrelse, såsom hypoplastiske underkæbe fænotyper 11. Zebrafisk embryoner tilbyder mange udviklingsmæssige og genetiske fordele for detaljeret undersøgelse af Meckel s brusk morfogenese. Deres genetiske sporbarhed, gennemsigtighed, ex vivo og hurtig udvikling er stærke fordele udlån det godt for observation af celle bevægelser og organisation ved direkte billedvisning 6. Brug slægt-sporing værktøjer, såsom sox10: Kaede transgene linje, har vi og andre afgrænset de neurale crest oprindelsen af embryonale kraniofacial skelet 1,5. Using af sox10: ERT2-Cre med ubi: Zebrabow-M transgene linje, er det nu muligt at udforske detaljer om cellulære bevægelser under kraniofacial udvikling. Den Zebrabow-M, er en transgen linje manipuleret med ubiquitinpromotoren driver ekspressionen af forskellige fluoroforer, hver flankeret af lox-steder 8. Den Zebrabow-M standard fluorophor er rød, der udtrykker RFP. Efter induktion af Cre udtryk, den Zebrabow-M konstruere rekombinerer og celler udtrykker en kombination af forskellige fluoroforer (RFP, FFP og YFP) skaber flere spektral udtryk i embryoet. Alle datterceller, som deler fra de mærkede celler efter rekombinationsbegivenheden derefter klonalt mærket, således at cellepopulationer, der stammer fra forskellige sidestillede progenitorer klonalt mærket. Ved denne kloning cellemærkning kan celler proliferation og migration med klonal opløsning følges (figur 1 og 2).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Pronase</td>
			<td>Roche Life Sciences</td>
			<td>10165921001</td>
			<td>Prepare 500 &#956;L stock aliquots at 50mg/mL</td>
		</tr>
		<tr>
			<td>Methylcellulose</td>
			<td>Sigma-Aldrich</td>
			<td>M0262</td>
			<td></td>
		</tr>
		<tr>
			<td>PTU (N-Phenylthiourea)</td>
			<td>Sigma-Aldrich</td>
			<td>P7619</td>
			<td></td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>E10521</td>
			<td></td>
		</tr>
		<tr>
			<td>4-HydoxyTamoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>T176</td>
			<td></td>
		</tr>
		<tr>
			<td>24 x 60 coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-548-5P</td>
			<td></td>
		</tr>
		<tr>
			<td>18 x 18 coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-540A</td>
			<td></td>
		</tr>
		<tr>
			<td>25 x 25 coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-540C</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR5&#39;-TOPO TA Cloning Kit</td>
			<td>Life technologies&#160;</td>
			<td>K591-20</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR/D-TOPO Cloning Kit&#160;</td>
			<td>Life Technologies</td>
			<td>K2400-20</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR3&#39;-pA</td>
			<td>Tol2Kit</td>
			<td>302</td>
			<td></td>
		</tr>
		<tr>
			<td>pDEST</td>
			<td></td>
			<td></td>
			<td>Gift from Geoffrey Burns labs</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bright field microscope&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image processing software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of chondrocyte intercalation and directional proliferation via zebrabow clonal cell analysis in the embryonic meckel&#8217;s cartilage

Udvikling af de hvirveldyr kraniofaciale strukturer kræver præcis koordinering af celle migration, spredning, vedhæftning og differentiering. Mønster af Meckel s brusk, en første svælg bue derivat, involverer migrering af kranie neurale crest (CNC) celler og den gradvise partitionering, spredning og organisering af differentierede chondrocytter. Flere undersøgelser har beskrevet CNC migration under underkæben morfogenese, men detaljerne i, hvordan chondrocyterne opnår organisation i vækst og udvidelse af Meckel s brusk fortsat uklart. Den sox10 begrænset og kemisk induceret Cre-rekombinase-medieret rekombination frembringer permutationer af forskellige fluorescerende proteiner (RFP, YFP og FFP), hvorved der skabes en multi-spektral mærkning af progenitorceller og deres afkom, der afspejler forskellige klonpopulationer. Brug konfokal time-lapse fotografering, er det muligt at observere chondrocyterne behavieller under udviklingen af ​​zebrafisk Meckel brusk. Multispektral celle mærkning gør det muligt for forskerne at påvise udvidelse af Meckel s chondrocytter. Under udvidelse fase af Meckel s brusk, som en forløber underkæben, chondrocytter interkalerer at foretage udvidelse, da de stak på en organiseret encellede lag række. Svigt i denne organiserede interkalerende proces at mægle celle forlængelse giver den cellulære mekanistisk forklaring på hypoplastisk underkæbe, som vi observerer i mandibulære misdannelser.

Traditionelle brusk visualisering af hele mount alcianblåt pletter har været uvurderlig i at observere udviklingen Meckel brusk og almindeligt anvendt til at visualisere endelige cellulære organisation 12 (figur 1A). For yderligere at analysere udviklingslandene chondrocytter overarbejde, afstamning sporing hjælp sox10: Kaede transgene linier har gjort det muligt for os at studere celle migration, konvergens og udvidelse i levende fostre 2,12 (Figur 1B).</stron...

Watch this Scientific Journal Video about Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckelâ€™s Cartilage at JoVE.

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel&#8217;s Cartilage

Celle organisering af kraniofaciale knogler har længe været en hypotese, men aldrig direkte visualiseres. Multispektral cellemærkning og in vivo levende billeddannelse tillader visualisering af dynamiske celle adfærd i zebrafisk underkæbe. Her har vi detaljeret protokollen til at manipulere Zebrabow transgene fisk og direkte observere celle intercalation og morfologiske ændringer af chondrocytter i Meckel s brusk.

Visualisering af Chondrocyt Intercalation og Directional spredning via Zebrabow Klonal Cell Analysis i Embryonale Meckel s Brusk

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. ...

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