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 MeckelÃ¢â‚¬â„¢s Cartilage at JoVE.

The authors thank Alex Schier for kindly sharing the Zebrabow-M transgenic line, Geoffrey Burns for the pDEST vector and Renee Ethier-Daigle for excellent care of the fish facility and lines.
FUNDING:
We are grateful for generous funding support from NIDCR RO3DE024490 and Shriners Hospitals for Children (E.C.L.) and post-doctoral training fellowships from Shriners Hospitals for Children (L.R. and Y.K).

Massachusetts General Hospital Institutional Animal Care and Use Committee (IACUC) approved all procedures under protocol number #2010N000106. This is in compliance with the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) guidelines.
1. Reagents and Materials Preparation
<ol>
	<li>Prepare 1 L of 50X E3 embryo medium (See Table 1) and prepare 1 L of 1X E3 embryo medium (See Table 2).</li>
	<li>Prepare E3 embryo me...

<ol>
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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Hedgehog+signaling+from+neural+to+oral+epithelium+organizes+anterior+craniofacial+development.">Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development.</a> Development. 133, 1069-1077 (2006).</li><li>Dixon, M. J., Marazita, M. L., Beaty, T. H., Murray, 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=Cleft+lip+and+palate:+understanding+genetic+and+environmental+influences.">Cleft lip and palate: understanding genetic and environmental influences.</a> Nature reviews Genetics. 12, 167-178 (2011).</li><li>Gfrerer, L., Dougherty, M., Liao, E. 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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. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+cartilage+and+bone.">Development of cartilage and bone.</a> Methods in cell biology. 76, 415-436 (2004).</li><li>McCarthy, N., 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=Pdgfra+protects+against+ethanol-induced+craniofacial+defects+in+a+zebrafish+model+of+FASD.">Pdgfra protects against ethanol-induced craniofacial defects in a zebrafish model of FASD.</a> Development. 140, 3254-3265 (2013).</li></ol>

Alcian blue and photoconvertible transgenic lines as described above complements each other to define the intricate process of cartilage and bone development. However, live cellular migration and organization during organogenesis has long been hypothesized and indirectly demonstrated but never visualized. Zebrabow-M transgenic line coupled with a cartilage specific Cre permits simultaneous live observation of all these distinct events involved in bone and cartilage formation. This technique allows the study of lower jaw ...

Craniofacial development requires complex molecular, cellular and tissue interactions to drive cell proliferation, migration and differentiation1,2,3. This tightly regulated and complex process is subject to genetic and environmental perturbations, such that craniofacial deformities are amongst the most common birth malformations1-9. While surgical interventions remain the mainstay of treatment for craniofacial anomalies, understanding the development basis is essential to innovate future therapies. Therefore, studying the morphogenesis and the mechanisms in the convergence and extension and cell integration provides novel insights into the formation of the craniofacial skeleton1.
Cranial neural crest migrate and populate the first pharyngeal arch, then form paired mandibular processes that extend to form the Meckel&#8217;s cartilage, which prefigures the mandible. Morphogenesis of the Meckel&#8217;s cartilage requires chondrocyte organization via directional proliferation, cell polarization and differentiation1,10. However, the intricacy of chondrocyte organization in the growth and extension of the Meckel&#39;s cartilage&#160;remains unclear. Understanding dynamic cell behavior&#160;is critical to understanding congenital malformations affecting mandibular size, such as hypoplastic mandible phenotypes11.
Zebrafish embryos offer many developmental and genetic advantages for detailed study of Meckel&#8217;s cartilage morphogenesis. Their genetic tractability, transparency, ex vivo and rapid development are powerful advantages lending it well for observation of cell movement and organization by live imaging6. Using lineage-tracing tools, such as sox10:kaede transgenic line, we and others have delineated the neural crest origins of the embryonic craniofacial skeleton1,5. Using the sox10:ERT2-Cre with the ubi:Zebrabow-M transgenic line, it is now possible to explore details of cellular movements during craniofacial development. The Zebrabow-M, is a transgenic line engineered with the ubiquitin promoter driving the expression of different fluorophores, each flanked by Lox sites8. The Zebrabow-M default fluorophore is Red, expressing RFP. After induction of Cre expression, the Zebrabow-M construct recombines and cells express a combination of different fluorophores (RFP, CFP and YFP) creating multi-spectral expression in the embryo. All the daughter cells that divide from the labeled cells after the recombination event are then clonally labeled, so that cell populations that derive from different juxtaposed progenitors are clonally labeled. By this cloning cell labeling, cells proliferation and migration with clonal resolution can be followed (Figure 1 and 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

Development of the vertebrate craniofacial structures requires precise coordination of cell migration, proliferation, adhesion and differentiation. Patterning of the Meckel&#39;s cartilage, a first pharyngeal arch derivative, involves the migration of cranial neural crest (CNC) cells and the progressive partitioning, proliferation and organization of differentiated chondrocytes. Several studies have described CNC migration during lower jaw&#160;morphogenesis, but the details of how the chondrocytes achieve organization in the growth and extension of Meckel&#8217;s cartilage remains unclear. The sox10 restricted and chemically induced Cre recombinase-mediated recombination generates permutations of distinct fluorescent proteins (RFP, YFP and CFP), thereby creating a multi-spectral labeling of progenitor cells and their progeny, reflecting distinct clonal populations. Using confocal time-lapse photography, it is possible to observe the chondrocytes behavior during the development of the zebrafish Meckel&#8217;s cartilage.
Multispectral cell labeling enables scientists to demonstrate extension of the Meckel&#8217;s chondrocytes.&#160;During extension phase of the Meckel&#8217;s cartilage, which prefigures the mandible, chondrocytes intercalate to effect extension as they stack in an organized single-cell layered row. Failure of this organized intercalating process to mediate cell extension provides the cellular mechanistic explanation for hypoplastic mandible that we observe in mandibular malformations.

Traditional cartilage visualization by whole mount Alcian blue stains has been invaluable in observing the developing Meckel&#8217;s cartilage and commonly used to visualize final cellular organization12 (Figure 1A). To further analyze the developing chondrocytes overtime, lineage tracing using sox10:Kaede transgenic lines has enabled us to study cell migration, convergence and extension in live embryos2,12(Figure 1B). However, the organization of chondrocy...

Watch this Scientific Journal Video about Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic MeckelÃ¢â‚¬â„¢s Cartilage at JoVE.

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic MeckelÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Cartilage

Cell organization of craniofacial bones has long been hypothesized but never directly visualized. Multi-spectral cell labeling and in vivo live imaging allows visualization of dynamic cell behavior in zebrafish lower jaw. Here, we detail the protocol to manipulate Zebrabow transgenic fish and directly observe cell intercalation and morphological changes of chondrocytes in the Meckel&#8217;s cartilage.

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

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

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