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 medium with N-Phenylthiourea (PTU) by adding 30 mg of PTU to 1 L of 1X E3. Stir O/N to ensure that the PTU has completely dissolved. Store at RT.</li>
	<li>To prepare pronase solution, dilute 150 &#181;l&#160;of pronase (0.5 mg/ml) with 15 ml&#160;of 1X E3. This amount is sufficient for 1 petri dish. Use immediately.</li>
	<li>Prepare Methylcellulose solution.
	<ol>
		<li>Prepare E3-tricaine solution by mixing 75 ml&#160;of 0.2% tricaine with 25 ml&#160;of 1X E3. Boil 3 g of methylcellulose in the 75 ml&#160;of E3-tricaine solution.</li>
		<li>Make up the final volume to 100 ml&#160;with E3-tricaine solution. The final methylcellulose solution is viscous and opaque with a yellowish tint. Storage: RT protected from the light.</li>
	</ol>
	</li>
	<li>Tamoxifen stock and induction solution
	<ol>
		<li>Prepare 5 mM tamoxifen stock solution by dissolving 4-hydroxytamoxifen in 100% ethanol. Store stock at -80 &#176;C.</li>
		<li>Prepare fresh dilution of tamoxifen at the desire concentration in 1X E3 to obtain the induction solution. CAUTION! Please consult MSDS before use.</li>
	</ol>
	</li>
	<li>Prepare 1 L of 0.2% tricaine as per Table 3.</li>
	<li>Obtain confocal slides. Glue four coverglass (18 x 18) together to form a block then place 2 blocks on a 24 x 60 coverglass 2 cm apart to allow mounting of the embryo in the middle.</li>
</ol>
2. Embryo Preparation
<ol>
	<li>Transgenic lines. 
	NOTE: Zebrabow-M was a generous gift from Alex Schier&#8217;s laboratory.</li>
	<li>sox10: ERT2-Cre 
	NOTE: Generate the targeting vector pDestTol2-sox10:ERT2-Cre using standard molecular cloning methods and commercially obtained recombination cloning technology.
	<ol>
		<li>Combine 5&#8217; entry clone (pENTR5&#8217;-sox10p) containing 7.2Kb of the sox10 promoter, a Gateway middle clone (pME-ERT2-Cre), and a 3&#8217; entry clone (pENTR3&#8217;-polyA) in an LR reaction with lens destination vector pDestTol2-&#945;-crystallin:YFP.</li>
		<li>Purify construct pDestTol2-sox10:ERT2-Cre. Co-inject the purified construct (100 ng/&#181;l) with Tol2 transposase mRNA (50 ng/&#181;l) into WT embryos (F0) at the one cell stage.</li>
		<li>Screen F0 adult founders for germ-line transmission based on strong yellow fluorescence in F1 embryos.</li>
	</ol>
	</li>
	<li>sox10: ERT2-Cre;Zebrabow-M
	<ol>
		<li>Outcross the Zebrabow-M line (2.1.1) with the sox10:ERT2-Cre transgenic line.</li>
		<li>Select embryos exhibiting a strong ubiquitous RFP expression and YFP lens marker and grow them till adults.</li>
	</ol>
	</li>
</ol>
3. Embryo Collection
<ol>
	<li>Set up several pairs of sox10:ERT2-Cre;Zebrabow-M transgenic zebrafish between 5 pm and 6 pm on Day 1.</li>
	<li>The following day, pull the dividers in the morning and collect the eggs around noon.</li>
	<li>Transfer them to petri dishes and add 15 ml&#160;of the pronase solution to dechorionate the embryos.</li>
	<li>Incubate the embryos for 1 to 5 min&#160;until approximately 60% of the embryos have been dechorionated. Stop the reaction by decanting the pronase solution and wash the embryos 3-4 times with fresh E3 medium.</li>
	<li>Incubate the dechorionated embryos in E3 at RT O/N and let them develop to reach the 10 somites stage. Incubate 50-60 embryos per clutch to avoid different developmental growth.</li>
</ol>
4. Treatment
<ol>
	<li>Check the embryos&#8217; developmental stage under a light field microscope to ensure they are at the 10 somites stage.</li>
	<li>Using a fluorescent microscope with a red fluorescent protein (RFP) filter, isolate the bright red embryos.</li>
	<li>Proceed to the induction of the Cre-recombination by adding 10 &#181;M of hydroxytamoxifen solution to the petri dish and incubating the isolated embryos at 28.5 &#176;C for 3 hr.</li>
	<li>Decant the tamoxifen solution and wash the embryos several times with E3. 
	CAUTION! discard the tamoxifen solution in a special biological waste container.</li>
	<li>Incubate the embryos at 28.5 &#176;C O/N.</li>
	<li>When the embryos are approximately the 28-29 somites stage (around 24 hr post fertilization), incubate the embryos at 28.5 &#176;C in E3-PTU solution hereafter. 
	NOTE: PTU is here used to inhibit formation of melanophores in the developing embryos. This treatment is necessary to avoid the signal distortion of fluorescence by auto-fluorescent melanophores.</li>
	<li>Incubate the embryos at 28.5 &#176;C</li>
</ol>
5. Embryo Selection
<ol>
	<li>To visualize the fully developed craniofacial structures, select the embryos at 4 days post fertilization for the intensity of the Red signal and Cre expression marker positivity (yellow in the retina).</li>
	<li>Anesthetize the embryos with E3/0.015% tricaine solution. Anesthesia is confirmed when no active movements or fin activity is observed when gently prodding the embryo.</li>
</ol>
6. Mounting the Embryo in Methycellulose
<ol>
	<li>Transfer the selected embryo for imaging into a petri dish containing a drop of methylcellulose and gently embalm the embryo with methylcellulose.</li>
	<li>On a clean confocal slide, place a drop of fresh methylcellulose and mount your embryo within the drop using a microloader tip. Position embryo dorsally taking caution not to touch the embryo directly.</li>
	<li>Cover the mounted embryo with a 25 x 25 coverslip and adjust the position of the embryo if necessary by manipulating the coverslip.</li>
	<li>The embryo is now ready to image.</li>
</ol>
7. Analysis by Fluorescence Microscopy
<ol>
	<li>Use the 20x dry lens objective (numerical aperture 0.75; pinhole size 2.0 &#181;m) for focusing the embryo.</li>
	<li>Press the L100 button on the microscope and select the channels in the software.</li>
	<li>Select the confocal setup button and click &#8216;auto&#8217; and select the following channels before closing the window: Ch1: CFP, Ch2: GFP, Ch3: RFP or mCherry</li>
	<li>Turn on Ch2 and Ch3 before clicking the &#8216;remove interlock&#8217; button. Click &#8216;play&#8217; to begin scanning.</li>
	<li>Focus on structure of interest, which is best done by starting with &#189; frame/sec and high voltage (HV) 150 and then adjusting the settings accordingly until the desired image quality is achieved. The RFP would be brighter than other colors, therefore adjust the HV. Change the Z position to find both YFP and RFP positive cells.</li>
	<li>Once set with the settings, capture the image of your embryo. Save your picture in the confocal imaging software format and TIFF format for image processing in each split channel.</li>
	<li>Next, for CFP imaging, turn off Ch2 and Ch3 and turn on Ch1. Re-adjust the settings as in step 5 without changing the focus area. 
	NOTE: CFP can be really hard to detect due to background signal in blue channel that can be circumvented by adjusting the pinhole size. A larger pinhole size permits better detection of the fluorescence by reducing signal/background noise ratio. However, a larger pinhole will reduce confocal effect, compromising image quality. Therefore, careful adjustment of the settings is required to achieve the desired image quality and intensity.</li>
	<li>Capture the image and save it in the both format (confocal software and TIFF) for image processing. Images can then be processed using common image processing software to merge layers and improve color settings as described by Pan and colleagues 8.</li>
</ol>

<ol>
	<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. 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. 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> Journal of visualized experiments : JoVE. , e50525 (2013).</li><li>McCollum, C. W., Ducharme, N. A., Bondesson, M., Gustafsson, 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=Developmental+toxicity+screening+in+zebrafish.+Birth+defects+research.">Developmental toxicity screening in zebrafish. 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>

The authors declare that they have no competing financial interests.

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><tbody><tr><td>Pronase</td><td>Roche Life Sciences</td><td>10165921001</td><td>Prepare 500 &amp;mu;l stock aliquots at 50 mg/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&#039;-TOPO TA Cloning Kit</td><td>Life technologies&amp;nbsp;</td><td>K591-20</td><td></td></tr><tr><td>pENTR/D-TOPO Cloning Kit&amp;nbsp;</td><td>Life Technologies</td><td>K2400-20</td><td></td></tr><tr><td>pENTR3&#039;-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>Bright field microscope&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Fluorescent microscope&amp;nbsp;</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 Meckelâ€™s Cartilage at JoVE.

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

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

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Research

JoVE Journal

Developmental Biology

9.0K Views.  Harvard Medical School. 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’s cartilage.

Video: Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel’s Cartilage

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Co-localization of Cell Lineage Markers and the Tomato Signal

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the reporter in a specific cell line and all progeny. Here, we used the cell lineage tracing technique to demonstrate that chondrocytes directly transform into osteoblasts and osteocytes during long bone and mandibular condyle development using two kinds of Cre, Col10a1-Cre and Aggrecan-CreERT2 (Agg-CreERT2), crossed with Rosa26tdTomato. Both Col10 and aggrecan are well-recognized markers for chondrocytes.
On this basis, we developed a new method-cell lineage tracing in conjunction with fluorescent immunohistochemistry-to define cell fate by analyzing the expression of specific cell markers. Runx2 (a marker for early-stage osteogenic cells) and Dentin matrix protein1 (DMP1; a marker for late-stage osteogenic cells) were used to identify chondrocyte-derived bone cells and their differentiation status. This combination not only broadens the application of cell lineage tracing, but also simplifies the generation of compound mice. More importantly, the number, location, and differentiation statuses of parent cell progeny are displayed simultaneously, providing more information than cell lineage tracing alone. In conclusion, the co-application of cell lineage tracing techniques and immunofluorescence is a powerful tool for investigating cell biology in vivo.

We developed two sets of tracing combinations in Rosa26tdtomato (ubiquitously expressed in all cells)/Cre (specifically expressed in chondrocytes) mice: one with 2.3Col1a1-GFP (specific to osteoblasts) and one with immunofluorescence (specific to bone cells). The data demonstrate the direct transformation of chondrocytes into bone cells.

The cell lineage tracing system has been used predominantly in developmental biology studies. The use of Cre recombinase allows for the activation of the ...

Dissection and Lateral Mounting of Zebrafish Embryos: Analysis of Spinal Cord Development

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Developmental processes such as proliferation, patterning, differentiation, and axon guidance can be readily modeled in the zebrafish spinal cord. In this article, we describe a mounting procedure for zebrafish embryos, which optimizes visualization of these events.

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene ...

Neuroscience

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its longevity can be a powerful way to reveal mechanisms of tissue development and maintenance. One of the most important tools currently available to monitor cells in vivo is the Confetti mouse model. The Confetti model can be used to genetically label individual cells in living mice with various fluorescent proteins in a cell type-specific manner and monitor their fate, as well as the fate of their progeny over time, in a process called clonal genetic tracing or clonal lineage tracing. This model was generated almost a decade ago and has contributed to an improved understanding of many biological processes, particularly related to stem cell biology, development, and renewal of adult tissues. However, preserving the fluorescent signal until image collection and simultaneous capturing of various fluorescent signals is technically challenging, particularly for mineralized tissue. This publication describes a step-by-step protocol for using the Confetti model to analyze growth plate cartilage that can be applied to any mineralized or nonmineralized tissue.

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Three and Four-Dimensional Visualization and Analysis Approaches to Study Vertebrate Axial Elongation and Segmentation

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a wide range of techniques encompassing ex vivo and in vitro approaches. However, most studies still rely on the analysis of two-dimensional (2D) imaging data, which limits proper evaluation of a developmental process like axial extension and somitogenesis involving highly dynamic interactions in a complex 3D space. Here we describe techniques that allow mouse live imaging acquisition, dataset processing, visualization and analysis in 3D and 4D to study the cells (e.g., neuromesodermal progenitors) involved in these developmental processes. We also provide a step-by-step protocol for optical projection tomography and whole-mount immunofluorescence microscopy in mouse embryos (from sample preparation to image acquisition) and show a pipeline that we developed to process and visualize 3D image data. We extend the use of some of these techniques and highlight specific features of different available software (e.g., Fiji/ImageJ, Drishti, Amira and Imaris) that can be used to improve our current understanding of axial extension and somite formation (e.g., 3D reconstructions). Altogether, the techniques here described emphasize the importance of 3D data visualization and analysis in developmental biology, and might help other researchers to better address 3D and 4D image data in the context of vertebrate axial extension and segmentation. Finally, the work also employs novel tools to facilitate teaching vertebrate embryonic development.

Here, we describe computational tools and methods that allow visualization and analysis of three and four-dimensional image data of mouse embryos in the context of axial elongation and segmentation, obtained by in toto optical projection tomography, and by live imaging and whole-mount immunofluorescence staining using multiphoton microscopy.

Somitogenesis is a hallmark of vertebrate embryonic development. For years, researchers have been studying this process in a variety of organisms using a ...

A Label-free Xenograft Model for Investigating the Behavior of Human Stem Cell Spheroids in Chick Embryos

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell migration, differentiation, and morphogenesis, with unique instructive signals and germ layer identity that are often absent from adult xenograft models. In addition, embryonic models cannot discriminate self versus non-self tissues, eliminating the risk of rejection of the graft and the need for immune suppression of the host. This paper presents a methodology for transplantation of spheroids of human cells into chicken embryos, which are accessible, amenable to manipulation, and develop at 37 &#176;C. 
Spheroids allow the selection of a specific region of the embryo for transplantation. After being grafted, the cells become integrated into the host tissue, allowing the follow-up of their migration, growth, and differentiation. This model is flexible enough to allow the utilization of different adherent populations, including heterogeneous primary cell populations and cancer cells. To circumvent the need for prior cell labeling, a protocol for the identification of donor cells through hybridization of human-specific Alu probes is also described, which is particularly important when investigating heterogeneous cell populations. Furthermore, DNA probes can be easily adapted to identify other donor species. This protocol will describe the general methods for preparing spheroids, grafting into chicken embryos, fixing and processing tissue for paraffin sectioning, and finally identifying the human cells using DNA in situ hybridization. Suggested controls, examples of interpretation of results and various cell behaviors that can be assayed will be discussed in addition to the limitations of this method.

Here, we describe a method to transplant and identify human cell spheroids into chick embryos. This xenograft model uses the embryonic microenvironment as a source of instructive signals to assay cell migration, differentiation, and tropism and is especially suited for the study of primary and/or heterogeneous cell populations.

Xenografts are valuable methods to investigate the behavior of human cells in vivo. In particular, the embryonic environment provides cues for cell ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

Bioengineering

In Vitro Preparation of Actively Maturing Bovine Articular Cartilage Explants for X-Ray Phase Contrast Imaging

Understanding the mechanisms that underpin post-natal maturation of articular cartilage is of crucial importance for designing the next generation of tissue engineering strategies and potentially repairing diseased or damaged cartilage. In general, postnatal maturation of the articular cartilage, which is a wholesale change in collagen structure and function of the tissue to accommodate growth of the organism, occurs over a timescale ranging from months to years. Conversely dissolution of the structural organization of the cartilage that also occurs over long timescales is the hallmark of tissue degeneration. Our ability to study these biological processes in detail have been enhanced by the findings that growth factors can induce precocious in vitro maturation of immature articular cartilage. The developmental and disease related changes that occur in the joint involve bone and cartilage and an ability to co-image these tissues would significantly increase our understanding of their intertwined roles.
The simultaneous visualization of soft tissue, cartilage and bone changes is nowadays a challenge to overcome for conventional preclinical imaging modalities used for the joint disease follow-up. Three-dimensional X-ray Phase-Contrast Imaging methods (PCI) have been under perpetual developments for 20 years due to high performance for imaging low density objects and their ability to provide additional information compared to conventional X-ray imaging.
In this protocol we detail the procedure used in our experiments from biopsy of the cartilage, generation of in vitro matured cartilage to data analysis of image collected using X-ray phase contrast imaging.

This protocol describes the preparation of in vitro bovine articular cartilage for imaging in high resolution with X-rays. These explants actively undergo postnatal maturation. We describe here the necessary steps from the biopsy to data analysis of 3D X-ray phase contrast imaging, passing through explant culture, tissue fixation and synchrotron preparation.

Understanding the mechanisms that underpin post-natal maturation of articular cartilage is of crucial importance for designing the next generation of ...

Optical Sectioning and Visualization of the Intervertebral Disc from Embryonic Development to Degeneration

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To decode disc degeneration and to be able to develop regenerative approaches a thorough understanding of the cellular biology of the IVD is essential. One aspect of this biology that still remains unanswered is the question of how cells are spatially arranged in a physiological state and during degeneration. The biological properties of the IVD and its availability make this tissue difficult to analyze. The present study investigates spatial chondrocyte organization in the anulus fibrosus from early embryonic development to end-stage degeneration. An optical sectioning method (Apotome) is applied to perform high resolution staining analyses using bovine embryonic tissue as an animal model and human disc tissue obtained from patients undergoing spine surgery. From a very high chondrocyte density in the early embryonic bovine disc, the number of cells decreases during gestation, growth, and maturation. In human discs, an increase in cellular density accompanied the progression of tissue degeneration. As had already been demonstrated in articular cartilage, cluster formation represents a characteristic feature of advanced disc degeneration.

We present a method to investigate spatial chondrocyte organization in the anulus fibrosus of the intervertebral disc using an optical sectioning method.

Intervertebral disc (IVD) degeneration is a leading cause of low back pain and it entails a high degree of impairment for the affected individuals. To ...

Visualization of Chondrocyte Intercalation and Directional Proliferation via Zebrabow Clonal Cell Analysis in the Embryonic Meckel’s Cartilage

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