We are grateful to the King&#8217;s College London Biological Services Unit, especially Tiffany Jarvis and Lynsey Cashnella for their continued support. We thank Derek Stemple, Mamoru Ishii and Robert Maxson for advice and help with reagents during initial establishment of this protocol. We thank Dheraj Taheem for help with gamma-irradiation of STO cells. We thank the Liu and Krause labs, especially Tommy Pallett, for great support. This work was funded by grants from the BBSRC (BB/R015953/1 to KJL/MK), a studentship from the MRC Doctoral Training Programme (LD), Newland Pedley Fund (ALM) and KRIPIS II, General Secretariat of Research and Technology (GSRT), Ministry of Education and Religious Affairs, Greece and Fondation Sant&#233; (to SGGM).

All animal work has undergone ethical approval by the King&#8217;s College London Ethical Review Process and was performed in accordance with UK Home Office Project License P8D5E2773 (KJL).
1. Preparation of reagents
<ol>
	<li>Prepare general solutions and tools including sterile phosphate buffer saline (PBS), 70% ethanol, dissection tools (forceps and dissection blades or sterile needles), plastic plates or glass slides coated with a commercially available extracellular matrix (ECM)-based hydrogel or fibronectin (see the Table of Materials), and neural crest media (see below).</li>
	<li>Prepare the neural crest basal medium using Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM, 4500 mg/L glucose), 15% fetal bovine serum (FBS), 0.1 mM minimum essential medium nonessential amino acids (MEM NEAA 100X), 1 mM sodium pyruvate, 55 &#181;M &#946;-mercaptoethanol, 100 units/mL penicillin, 100 units/mL streptomycin, and 2 mM L-glutamine.
	<ol>
		<li>Condition the media overnight using growth-inhibited STO feeder cells21.
		<ol>
			<li>Prepare STO cells (see the Table of Materials) media to contain DMEM supplemented by 10% FBS and 100 U/mL penicillin, 100 U/mL streptomycin. Grow and expand STO cells to confluence in 25 cm2 flasks coated with 0.1% gelatin. Apply 5000 rad of gamma irradiation.</li>
			<li>Seed approximately 3 x 106 growth-inhibited cells onto a 10 cm2 dish or 25 cm2 flask (from step 1.2.1.1). Add approximately 10&#8211;12 mL of neural crest basal medium and incubate overnight. 
			NOTE: Seeded cells can be used to produce conditional medium for up to 10 days. Check appearance of cells regularly</li>
		</ol>
		</li>
		<li>Filter the medium (0.22 &#181;m pore size), and supplement with 25 ng/mL basic fibroblast growth factor (bFGF) and 1000 U of leukemia inhibitor factor (LIF). 
		NOTE: Store at 4 &#176;C and use within a month or store at -20 &#176;C and use within 3 months.</li>
	</ol>
	</li>
	<li>Coat the tissue culture surfaces with extracellular matrix. 
	NOTE: Depending on the biological question being asked, the matrix can be coated onto glass-bottomed culture dishes, plastic tissue culture dishes or glass cover slips. See below for differing ECM based hydrogel dilutions dependent on the matrix substrate. Fibronectin has been tested on glass-bottomed dishes and cover slips only at the concentrations specified below. Here on, we will refer to the substrate-coated surfaces as &#8220;coated plates&#8221;.
	<ol>
		<li>Coat the tissue culture surfaces with ECM based hydrogel. 
		NOTE: Keep the substrate cold until plating, either by cooling the media or keeping on ice.
		<ol>
			<li>Thaw the hydrogel at 4 &#176;C overnight. Add 5 mL of 10% FBS in DMEM to 5 mL of hydrogel for a final volume of 10 mL (see the Table of Materials).</li>
			<li>Make 0.5&#8211;1 mL aliquots as convenient and store at -20 &#176;C.</li>
			<li>Thaw the hydrogel aliquots on ice.</li>
			<li>Use a 1:20 dilution of the hydrogel stock to coat plastic.</li>
			<li>Use a 1:5 dilution of the hydrogel stock to coat glass slides and glass-bottomed tissue culture plates. 
			NOTE: Dilute the hydrogel in cold DMEM.</li>
			<li>Apply enough diluted hydrogel to cover the desired area on to plates/slides and incubate for 30&#8211;45 min at 37 &#176;C.</li>
			<li>Use coated plates/slides immediately or store coated slides at 4 &#176;C overnight.</li>
			<li>Remove excesses and rinse slides with high glucose DMEM (optional) before use.</li>
		</ol>
		</li>
		<li>Coat the tissue culture surfaces with fibronectin.
		<ol>
			<li>Make aliquots of 1 mg/mL fibronectin stock solution and store at -80 &#176;C. Dilute fibronectin with Dulbecco&#8217;s PBS (dPBS) to a final concentration of 1 &#181;g/mL.</li>
			<li>Apply sufficient fibronectin to cover the desired area and incubate at room temperature for 15&#160;min.</li>
			<li>Remove residual fibronectin and allow the glass to dry for 30-45 min.</li>
			<li>Rinse wells or cover slips with high glucose DMEM (optional) before use.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Day 1: Dissection of early somite stage embryos
NOTE: Use sterile tools and sterile solutions. If genotyping is needed, collect the body of the embryo for DNA extraction.
<ol>
	<li>Dissection of the cranial neural plate is restricted to embryos at 8.5 days post coitum (dpc). Select embryos&#160;at the 5&#8211;8 somite stage. Dissect the uterus into PBS and cut the mesometrium to separate each embryo (Figure 1A). The muscular wall of the uterus contracts and the decidual tissue will become visible (Figure 1B). 
	NOTE: Maintain embryos in the uterus in ice-cold PBS while dissections are performed one embryo at a time. Move embryos with a glass Pasteur pipette into fresh sterile PBS to improve visibility and reduce contamination.</li>
	<li>Slide forceps between the muscle layer and decidual tissue and remove the muscle layer with a second pair of forceps (Figure 1C).</li>
	<li>Using forceps, pierce the deciduum at the edges of the mesometrial pole and with a second pair of forceps tear to open perpendicularly to the pole.</li>
	<li>Peel back the decidual tissue with the forceps to visualize the Reichert&#8217;s membrane.</li>
	<li>Remove Reichert&#8217;s membrane carefully. The visceral yolk sac becomes visible and the embryo can be seen inside (Figure 1D).</li>
	<li>Remove the visceral yolk sac and the amnion (Figure 1E) and position the embryo in order to visualize the head fold (Figure 1F).</li>
	<li>Cut the head fold above the heart and scrape away the underlying mesoderm using forceps and/or eyelash tools to obtain a clean neural plate (NP) (Figure 1H). 
	NOTE: The NP can be kept whole or divided down the anteroposterior axis so that each side can be plated individually. The neural plate border can be further trimmed away from the neural plate in order to minimize neuronal contributions to the explants.</li>
	<li>Use a glass Pasteur pipette to transfer the dissected neural plate onto a hydrogel-coated dish filled with conditioned neural crest media.</li>
	<li>Gently swirl the dish to position the NP in the middle of the well. This is important for maximizing the phase quality for live-cell imaging (on day 2).</li>
	<li>Incubate overnight (or to desired time-point) at 37 &#176;C in 5% CO2. Neural crest cells should be visibly migrating out of the neural plate. 
	NOTE: Cells usually attach within 6&#8211;8 h. After the explant attaches, allow more time to visualize migrating cells. Usually by 24 h post explant, we can find three distinguishable populations of cells. The first population, at the center of the explant, is the neural plate (NP). The second population, the premigratory NC (pNC), surrounds the NP in an epithelial sheet of cells. The third population, in the outside ring, is formed of migratory NC (mNC), which are bigger in size, and appear fully mesenchymal (Figure 2).</li>
</ol>
3. Day 2: Live cell imaging of murine cranial neural crest cells
NOTE: Imaging should be performed at 24 h post explanting to optimally image and quantify neural crest cell migration. NC induction media does not need to be refreshed before live cell imaging. Access to an inverted microscope, with a motorized stage and an incorporated environment chamber is required. Use multi-well tissue culture dishes suitable for imaging (Table of Materials).
<ol>
	<li>Microscope set-up
	<ol>
		<li>Set the environment chamber at 37 &#176;C and 5% CO2.</li>
		<li>Pierce a hole into the lid of the tissue culture plate lid to allow the CO2 needle, connected to the CO2 humidification chamber, to sit within the plate.</li>
		<li>Place the tissue culture dish into the specimen holder and tape down the plate lid and CO2 needle to prevent shaking during multi-well acquisition.</li>
		<li>Switch on the microscope controller, the stage controller and the imaging software.</li>
		<li>Focus on the cranial NC cells at 10x magnification (with matching phase ring in the condenser selected).</li>
		<li>Set high quality phase-contrast on the microscope by adjusting the field iris diaphragm, aperture iris diaphragm and centering telescope, as specified in the microscope set-up manual.</li>
	</ol>
	</li>
	<li>Phase contrast live-cell imaging
	<ol>
		<li>Set the directory or file location where the time-lapse files will be saved.</li>
		<li>Set the exposure time, binning, and camera area.</li>
		<li>Set the number of time points, duration of imaging and time interval between frames.
		<ol>
			<li>To quantify NC cell migratory capacity, set the microscope to 10x magnification, taking 1 frame every 5 min (217 time points over 18 h). To quantify cell morphology, set magnification to 40x, taking 1 frame/min (61 time points over 1 h). To quantify lamellipodial dynamics, set magnification to 40x or 60x magnification, taking 1 frame every 10 s (over 10 min).</li>
		</ol>
		</li>
		<li>For multi-well imaging, set the mechanical stage to move between selected XY positions of interest. Confirm that the cranial NC cells are in focus and the stage positions are correct.</li>
		<li>Use the Acquire command to start time-lapse imaging.</li>
		<li>Once time-lapse imaging is complete, review the multidimensional data and export .stk files for analysis. 
		NOTE: .stk is a TIFF stack file.</li>
		<li>Exit the software, shut down the computer and turn off the stage, camera and microscope controllers.</li>
	</ol>
	</li>
</ol>
4. Imaging analysis: quantification of neural crest cell migration
NOTE: To better define the cellular behaviors exhibited by migrating murine cranial neural crest cells, we have analyzed a series of quantifiable migratory parameters, specifically focusing on migratory capacity and cell shape dynamics. (1) Migration (accumulated distance) is the total path length taken by the cell (&#181;m); (2) Migration (Euclidean distance) is the straight-line distance between initial and final position of cell (&#181;m); (3) Migration (cell speed) is distance traveled by cell per unit of time (&#181;m/min); (4) Cell Shape (cell area) is the total surface covered by cell. Set pixel to micron scale according to imaging microscope. (A = Apx x Npx, where Apx = pixel area and Npx = number of pixels. Units: &#181;m2; (5) Cell shape (cell circularity) is the deviation of cell shape from a perfect circle which is indicated by a circularity value of 1.0 (4&#960; (A/P2)) where A = area and P = perimeter.
<ol>
	<li>Single cell tracking 
	NOTE: To measure NC cell migration, XY coordinates of individual cells across all time-lapse frames are generated. This allows for subsequent analysis of distance, speed and persistence measures of cell migration.
	<ol>
		<li>Open ImageJ and import data as TIFF stack files.</li>
		<li>Click Analyze | Set Scale to calibrate the .stk files according to microscope settings, working in pixel/&#956;m.</li>
		<li>Click Plugins | Tracking | Manual Tracking to open Image J manual cell tracking plugin. To begin cell tracking, select Add track.</li>
		<li>Track cells through all frames of time-lapse movies, using the nucleus as a reference point. 
		NOTE: 10&#8211;20 cells should be tracked per explant, with a total of 60 cells tracked (n = 3). Cells that undergo cell division during the course of time-lapse should be excluded from analysis.</li>
		<li>Save and export the results as a .csv file. Results represent individual cell track number, slice number and XY coordinates over all frames.</li>
	</ol>
	</li>
	<li>Quantification of neural crest cell migratory capacity
	<ol>
		<li>Open single cell tracking data (see above). Convert .csv files into .txt file format.</li>
		<li>Open the migration software (the Table of Materials). Click the Import Data tab to import the cell tracking data as a .txt file.</li>
		<li>Under Datasets | Initialization, select the number of slices or frames to be analyzed and set the XY calibration and time interval between frames. Select Apply settings to save the settings.</li>
		<li>Select the Plot Data symbol to form trajectory plots. Select the Statistics symbol to quantify distance and speed measures.</li>
		<li>Save the trajectory plots as bitmap (.bmp) files, and distance and speed measures as .txt files. Select the Remove Data symbol. Repeat for other time-lapse files. 
		NOTE: Trajectory plots can be used to visualize the directness of individual cell paths for a given cell condition or state over the course of time-lapse movies (Figure 4A). Distance and speed data stored in the .txt files can then be used for further analysis.</li>
	</ol>
	</li>
	<li>Quantification of neural crest cell area and circularity
	<ol>
		<li>Open the time-lapse .stk files in ImageJ and calibrate according to microscope settings, working in pixel/&#181;m.</li>
		<li>Under Analyze | Set Measurements, click to select the cell shape parameters: cell area, perimeter and shape descriptor.</li>
		<li>Use the Freehand Selection tool&#160;to manually draw around each cell, using cell membrane boundaries as a guide.</li>
		<li>Press Ctrl + B keys on the keyboard to maintain the cell outline overlain on the image. Repeat for cells over each time-lapse frame.</li>
		<li>Use the Image | Overlay | To ROI Manager to store the values.</li>
		<li>Once all cells of interest per frame have been outlined, click Measure. Save the results as a .csv file. 
		NOTE: 10&#8211;20 cells per movie should be outlined, with a total of 30&#8211;60 cells analyzed per condition (n = 3). Cell shape data (.csv files) can be used to quantify how cell shape dynamics change over time (Figure 4C) or how morphology may be altered under different cell treatments.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Duband, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+in+the+molecular+and+cellular+strategies+of+epithelium-to-mesenchyme+transitions:+Insights+from+the+neural+crest.">Diversity in the molecular and cellular strategies of epithelium-to-mesenchyme transitions: Insights from the neural crest.</a> Cell Adhesion &amp; Migration. 4, 458-482 (2010).</li><li>Mayor, R., Carmona-Fontaine, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keeping+in+touch+with+contact+inhibition+of+locomotion.">Keeping in touch with contact inhibition of locomotion.</a> Trends in Cell Biology. 20, 319-328 (2010).</li><li>Le Douarin, N., Kalcheim, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The neural crest. 2nd edn. , (1999).</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=.">.</a> Neural Crest Cells : Evolution, Development, and Disease. , (2014).</li><li>Jiang, R., Bush, J. O., Lidral, A. C. <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+the+upper+lip:+morphogenetic+and+molecular+mechanisms.">Development of the upper lip: morphogenetic and molecular mechanisms.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 235, 1152-1166 (2006).</li><li>Ahola, J. 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=Increased+incidence+of+Hirschsprung's+disease+in+patients+with+hypoplastic+left+heart+syndrome--a+common+neural+crest-derived+etiology?.">Increased incidence of Hirschsprung's disease in patients with hypoplastic left heart syndrome--a common neural crest-derived etiology?.</a> Journal of Pediatric Surgery. 44, 1396-1400 (2009).</li><li>Bajpai, R., 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=CHD7+cooperates+with+PBAF+to+control+multipotent+neural+crest+formation.">CHD7 cooperates with PBAF to control multipotent neural crest formation.</a> Nature. 463, 958-962 (2010).</li><li>Inoue, 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=Congenital+hypomyelinating+neuropathy,+central+dysmyelination,+and+Waardenburg-Hirschsprung+disease:+phenotypes+linked+by+SOX10+mutation.">Congenital hypomyelinating neuropathy, central dysmyelination, and Waardenburg-Hirschsprung disease: phenotypes linked by SOX10 mutation.</a> Annals of Neurology. 52, 836-842 (2002).</li><li>Kim, H., Kang, K., Ekram, M. B., Roh, T. Y., Kim, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aebp2+as+an+epigenetic+regulator+for+neural+crest+cells.">Aebp2 as an epigenetic regulator for neural crest cells.</a> PloS one. 6, e25174 (2011).</li><li>Barriga, E. H., Trainor, P. A., Bronner, M., 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=Animal+models+for+studying+neural+crest+development:+is+the+mouse+different?.">Animal models for studying neural crest development: is the mouse different?.</a> Development. 142, 1555-1560 (2015).</li><li>Bronner, M. E., LeDouarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+evolution+of+the+neural+crest:+an+overview.">Development and evolution of the neural crest: an overview.</a> Developmental Biology. 366, 2-9 (2012).</li><li>De Calisto, J., Araya, C., Marchant, L., Riaz, C. F., 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=Essential+role+of+non-canonical+Wnt+signalling+in+neural+crest+migration.">Essential role of non-canonical Wnt signalling in neural crest migration.</a> Development. 132, 2587-2597 (2005).</li><li>Carmona-Fontaine, C., 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=Contact+inhibition+of+locomotion+in+vivo+controls+neural+crest+directional+migration.">Contact inhibition of locomotion in vivo controls neural crest directional migration.</a> Nature. 456, 957-961 (2008).</li><li>Matthews, H. 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=Directional+migration+of+neural+crest+cells+in+vivo+is+regulated+by+Syndecan-4/Rac1+and+non-canonical+Wnt+signaling/RhoA.">Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA.</a> Development. 135, 1771-1780 (2008).</li><li>Banerjee, 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=A+novel+role+for+MuSK+and+non-canonical+Wnt+signaling+during+segmental+neural+crest+cell+migration.">A novel role for MuSK and non-canonical Wnt signaling during segmental neural crest cell migration.</a> Development. 138, 3287-3296 (2011).</li><li>Pryor, S. E., 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=Vangl-dependent+planar+cell+polarity+signalling+is+not+required+for+neural+crest+migration+in+mammals.">Vangl-dependent planar cell polarity signalling is not required for neural crest migration in mammals.</a> Development. 141, 3153-3158 (2014).</li><li>Serbedzija, G. N., Bronner-Fraser, M., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vital+dye+analysis+of+cranial+neural+crest+cell+migration+in+the+mouse+embryo.">Vital dye analysis of cranial neural crest cell migration in the mouse embryo.</a> Development. 116, 297-307 (1992).</li><li>Kawakami, M., Umeda, M., Nakagata, N., Takeo, T., Yamamura, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+migrating+mouse+neural+crest+cell+assay+system+utilizing+P0-Cre/EGFP+fluorescent+time-lapse+imaging.">Novel migrating mouse neural crest cell assay system utilizing P0-Cre/EGFP fluorescent time-lapse imaging.</a> BMC Developmental Biology. 11, 68 (2011).</li><li>Stemple, D. L., Anderson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+stem+cell+for+neurons+and+glia+from+the+mammalian+neural+crest.">Isolation of a stem cell for neurons and glia from the mammalian neural crest.</a> Cell. 71, 973-985 (1992).</li><li>Etchevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+culture+of+chick,+mouse+or+human+neural+crest+cells.">Primary culture of chick, mouse or human neural crest cells.</a> Nature protocols. 6, 1568-1577 (2011).</li><li>Ishii, 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=A+stable+cranial+neural+crest+cell+line+from+mouse.">A stable cranial neural crest cell line from mouse.</a> Stem Cells and Development. 21, 3069-3080 (2012).</li><li>Gonzalez Malagon, S. G., Liu, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ALK+and+GSK3:+Shared+Features+of+Neuroblastoma+and+Neural+Crest+Cells.">ALK and GSK3: Shared Features of Neuroblastoma and Neural Crest Cells.</a> Journal of Experimental Neuroscience. 12, 1179069518792499 (2018).</li><li>Gonzalez Malagon, S. G., 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=Glycogen+synthase+kinase+3+controls+migration+of+the+neural+crest+lineage+in+mouse+and+Xenopus.">Glycogen synthase kinase 3 controls migration of the neural crest lineage in mouse and Xenopus.</a> Nature Communications. 9, 1126 (2018).</li><li>Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+gene+activity+in+mouse+embryos+in+utero+by+a+tamoxifen-inducible+form+of+Cre+recombinase.">Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase.</a> Current Biology : CB. 8, 1323-1326 (1998).</li><li>Laranjeira, C., 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=Glial+cells+in+the+mouse+enteric+nervous+system+can+undergo+neurogenesis+in+response+to+injury.">Glial cells in the mouse enteric nervous system can undergo neurogenesis in response to injury.</a> Journal of Clinical Investigation. 121, 3412-3424 (2011).</li><li>Pfaltzgraff, E. R., Mundell, N. A., Labosky, 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=Isolation+and+culture+of+neural+crest+cells+from+embryonic+murine+neural+tube.">Isolation and culture of neural crest cells from embryonic murine neural tube.</a> Journal Of Visualized Experiments : JoVE. , e4134 (2012).</li><li>Sternberg, J., Kimber, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relationship+between+emerging+neural+crest+cells+and+basement+membranes+in+the+trunk+of+the+mouse+embryo:+a+TEM+and+immunocytochemical+study.">The relationship between emerging neural crest cells and basement membranes in the trunk of the mouse embryo: a TEM and immunocytochemical study.</a> Journal of Embryology and Experimental Morphology. 98, 251-268 (1986).</li><li>Sternberg, J., Kimber, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distribution+of+fibronectin,+laminin+and+entactin+in+the+environment+of+migrating+neural+crest+cells+in+early+mouse+embryos.">Distribution of fibronectin, laminin and entactin in the environment of migrating neural crest cells in early mouse embryos.</a> Journal of Embryology and Experimental Morphology. 91, 267-282 (1986).</li><li>George, E. L., Georges-Labouesse, E. N., Patel-King, R. S., Rayburn, H., Hynes, R. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defects+in+mesoderm,+neural+tube+and+vascular+development+in+mouse+embryos+lacking+fibronectin.">Defects in mesoderm, neural tube and vascular development in mouse embryos lacking fibronectin.</a> Development. 119, 1079-1091 (1993).</li><li>Henderson, D. J., Copp, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+the+extracellular+matrix+in+neural+crest+cell+migration.">Role of the extracellular matrix in neural crest cell migration.</a> Journal of Anatomy. 191 (Pt 4), 507-515 (1997).</li><li>Thomas, 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=Human+neural+crest+cells+display+molecular+and+phenotypic+hallmarks+of+stem+cells.">Human neural crest cells display molecular and phenotypic hallmarks of stem cells.</a> Human Molecular Genetics. 17 (21), 3411-3425 (2008).</li><li>Greenberg, J. H., Seppa, S., Seppa, H., Hewitt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+collagen+and+fibronectin+in+neural+crest+cell+adhesion+and+migration.">Role of collagen and fibronectin in neural crest cell adhesion and migration.</a> Developmental Biology. 87 (2), 259-266 (1981).</li><li>Leung, A. W., 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=WNT/&#946;-catenin+signaling+mediates+human+neural+crest+induction+via+a+pre-neural+border+intermediate.">WNT/&#946;-catenin signaling mediates human neural crest induction via a pre-neural border intermediate.</a> Development. 143 (3), 398-410 (2016).</li><li>Zhu, Q., Lu, Q., Gao, R., Cao, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prospect+of+Human+Pluripotent+Stem+Cell-Derived+Neural+Crest+Stem+Cells+in+Clinical+Application.">Prospect of Human Pluripotent Stem Cell-Derived Neural Crest Stem Cells in Clinical Application.</a> Stem Cells International. 2016, 7695836 (2016).</li></ol>

The authors have no conflicts of interest.

Studying mammalian neural crest cells has been a challenge for scientists because of the in utero nature of mammalian development. In vivo studies are difficult to set up, as the embryo must be manipulated under conditions that mimic life in the uterus. In practice, it is nearly impossible to reproducibly culture these (E8+) embryos for longer than 24 h, especially for live imaging. Furthermore, neural crest induction and migration occur concurrently with neural tube closure and embryonic turning in the mouse; this is a crucial and stressful morphogenetic event, which frequently fails when embryos are cultured ex utero. Thus, the success rate of ex utero approaches is generally low. The use of immortalized NC cells21 is a useful tool to reduce animal use and it may provide a better source of neural crest cells for long-term analysis, transfection, and enrichment studies. However, there is clearly a need to reliably culture primary neural crest cells. Our method is applicable to mouse knock-out or conditional genetic models. A comparable method to ours has been described for other neural crest populations20; however, our method thoroughly describes the step-by-step isolation of murine cranial NC cells. We also describe the use of different matrices as well as the migration analysis procedure in detail.
To achieve consistent results, we found that special attention paid towards staging during the selection of embryos. Not surprisingly, the number of somites correlates with different stages in cranial NC development. Therefore, the knowledge of the embryo anatomy is very important before acquiring any experimental data. This approach can then be adapted towards isolating discrete populations of neural crest cells, depending on the biological question and the target cells.
Once the embryos are selected and dissected, mesodermal cells can be readily distinguished and should be removed to allow better visualization and to reduce contamination. For longer-term cultures, the neural plate tissue can be removed at 24 h of plating in order to prevent contamination by neural tissues. A further refinement could be the use of fluorescent lineage labeling (for example, using a Wnt1::cre or Sox10::creERT drivers combined with fluorescent reporters24,25) to distinguish neural crest cells from other tissues as shown in Figure 2C.
Previous reports have highlighted the potential of plating mouse NC explant cultures on different matrices, most commonly on commercial ECM hydrogels, fibronectin and collagen I20,21,26. In our hands, mouse cranial NC explant cultures are successfully grown on all three matrices, at concentrations specified in original reports (data not shown). The initial refined approach we adapted for our NC explant cultures used a commercial hydrogel as the matrix of choice, which primarily consisted of laminin and collagens21 (Figure 3A&#8211;B). However, composition of this hydrogel is not clearly defined, with unknown growth factor and protein content. As such, we have since shifted our approach to plating mouse NC explant cultures on fibronectin (Figure 3C&#8211;D). Fibronectin is well defined and highly expressed in the ECM and basement membranes along which NC cells migrate in vivo28,29,30. To optimize a fibronectin matrix that best replicates neural crest cell migration and morphology as seen using the hydrogel, we compared NC cell behaviors exhibited on the hydrogel against a titration of 0.25&#8211;30 &#181;g/mL fibronectin, and defined 1 &#181;g/mL fibronectin as providing ideal properties (data not shown). We believe that this preliminary work may help establish a framework for the systematic comparison of matrices, such as fibronectin, against those previously described, namely collagen and laminin32,33,34. It would be especially interesting to compare mouse NC cell migratory capacity on fibronectin versus collagen I, given that collagen-IA1 is endogenously secreted by mouse, avian and human NC cells28,30,31,32. Collagen I is therefore as relevant as fibronectin in the consideration of matrix choice. It is also worth acknowledging that the bioavailability of growth factors in the media may be altered by different matrix components, especially given the high serum content of our media. To overcome this, we are currently working to produce serum-free defined culture conditions. These defined media are successfully used in neural crest induction protocols in the pluripotent stem cell field, but require further optimization for our NC explant culture system33,34. Our work may also serve as a starting point for refining conditions for other types of NC cells such as cardiac and trunk NC, and for subsequent studies of NC differentiation. Most importantly, this protocol allows the isolation of cranial NC cells for a variety of applications. We envision studies on directed migration, 3-D migration and invasion. Cells isolated in this manner can be treated in vitro for a number of analyses. For example, cells can readily be treated using different small molecules to target specific proteins, they can be treated at defined time points, and washout experiments can be designed to determine recovery of cell behaviors (Figure 4). Longer term culture for transfection and differentiation assays is possible, as well as passage of cells (data not shown). However, the viability, the capacity of cell renewal and multipotency should be validated after passaging. Cells plated on glass coverslips can also be used in immunofluorescent staining protocols, following live imaging. Finally, this approach represents a tremendously powerful system for studying migration of NC from genetic mouse models22,23,24,25.

The neural crest (NC) lineage is a transient, multipotent and migratory population of cells that appears exclusively in vertebrates during early embryonic development1,2. Neural crest derivatives are extremely diverse, and include glia, smooth muscle, melanocytes, neurons and craniofacial bone and cartilage3,4. Because the neural crest contributes to the function of many organ systems, this lineage is essential for human embryogenesis. Aberrant NC development is implicated in a wide range of the most common human birth defects (i.e., cleft lip and palate)5, and also disorders such as Hirschsprung&#8217;s disease (HSCR), Wardensburg syndrome (WS), CHARGE syndrome and Williams Syndrome6,7,8,9.
NC development has been explored in a number of non-mammalian model systems including Xenopus, chick and zebrafish models. In mammals, work in mouse models has identified some of the key genetic events underlying neural crest development; however, it has been more difficult to follow the cell biology of neural crest migration, due to the inaccessibility of the mouse embryo (reviewed elsewhere10,11). Furthermore, while studies in chick, Xenopus and zebrafish have established a gene regulatory network for NC, loss of function studies in these animal models sometimes do not exhibit a comparable phenotype in mouse. For example, in Xenopus, zebrafish and chick, non-canonical Wnt signaling is one of the cellular mechanisms that allows the NC to acquire its migratory capacity12,13,14,15. However, in mouse, loss of non-canonical Wnt signaling does not seem to affect migration16. As in vivo NC migration has been difficult to track for long periods in mouse, it is unclear whether these species-differences reflect differing modes of migration, or differences in molecular regulation.
As noted, NC studies in mouse have been very challenging because the ex utero culture of embryos is laborious. Moreover, the NC is constantly in intimate contact with adjacent tissues such as mesoderm and neurectoderm. Recent use of neural crest-specific Cre drivers or exogenous dyes has allowed us to fluorescently label the migratory NC; however, these approaches are still limited. Despite multiple reports describing different techniques to visualize NC migration17,18, it has been difficult to resolve these techniques into a simple and routine procedure.
It is clear that there is a need for techniques that allow the handling and characterization of mammalian NC. We focused our efforts on the mouse cranial NC as it is the primary model for studying human craniofacial development and neurocristopathies. We refined our approach based on several interesting reports describing primary culture of NC cells19,20,21. Here, we thoroughly describe the optimal culture techniques for explanting primary NC cells. We demonstrate the live cell imaging method and the optimal use of different matrices to coat the culture plates. Our protocol describes how to capture the migration of live NC cells using an inverted microscope, which is intended as a guideline for use with other microscopes, as well as a detailed summary of our cellular analyses.
The expected result from the explant should be a beautifully laid out distribution of cells that are clearly distinguished under the microscope, where one can see three different populations of cells which represent (i) neural plate, (ii) premigratory, and, (iii) migratory neural crest cells. We demonstrate how to analyze the cell behaviors at the border of the premigratory population of cells during the epithelial-mesenchymal transition. We also focused our effort on studying fully migratory cells for cell speed, distance and cell morphology.

<table><tbody><tr><td>bFGF</td><td>R&amp;amp;D systems</td><td>233-FB</td><td></td></tr><tr><td>b-mercaptoethanol</td><td>Gibco</td><td>31350-010</td><td></td></tr><tr><td>Tissue culture flasks</td><td>Corning</td><td>430639</td><td>25cm2 culture flasks</td></tr><tr><td>DMEM (4500 mg/L glucose)</td><td>Sigma</td><td>D5671</td><td></td></tr><tr><td>Dulbecco&#039;s phosphate-buffered saline (dPBS)</td><td>Sigma</td><td>D8537</td><td></td></tr><tr><td>Ethanol</td><td>Fisher Chemicals</td><td>E/0650DF/C17</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Sigma</td><td>TFS AA 10-155</td><td></td></tr><tr><td>Fetal Bovine Serum (ES Cell FBS)</td><td>Gibco</td><td>26140079/16141-079</td><td></td></tr><tr><td>Fibronectin bovine plasma</td><td>Sigma</td><td>F1141</td><td></td></tr><tr><td>Gelatin from bovine skin</td><td>Sigma</td><td>G9382</td><td></td></tr><tr><td>L-glutamine</td><td>Sigma</td><td>G7513</td><td></td></tr><tr><td>Glass-bottomed, multi-well 24-well tissue culture plate</td><td>Ibidi</td><td>82406</td><td>Glass-bottomed, No 1.5, 24-well tissue culture dish with black sides</td></tr><tr><td>Cell migration analysis tool</td><td>Ibidi</td><td>v2.0</td><td>Manuals for this software can be found at: https://ibidi.com/manual-image-analysis/171-chemotaxis-and-migration-tool.html.</td></tr><tr><td>Circularity Plug-in</td><td>ImageJ</td><td>v1.29 or later</td><td>The circularity plug-in is an extended version of ImageJ/Fiji&amp;rsquo;s Measure command, designed by Rasband, W., (2000) (wsr@nih.gov).</td></tr><tr><td>LIF</td><td>ESGRO by Millipore</td><td>ESG1106</td><td></td></tr><tr><td>Manual Cell Tracking Plug-in</td><td>ImageJ</td><td>v1.34k or later</td><td>ref Cordelieres F. Institut Curie, Orsay (France) 2005</td></tr><tr><td>Microscope Image Analysis Software</td><td>Universal Imaging Corporation</td><td>6.3r7</td><td>MetaMorph Software Series 6.3r7</td></tr><tr><td>MEM non-essential aminoacids 100X</td><td>Gibco</td><td>11140-050</td><td></td></tr><tr><td>Matrigel (ECM-based Hydrogel)</td><td>Corning</td><td>356234</td><td></td></tr><tr><td>Microscope</td><td>Olympus</td><td>1X81</td><td>Olympus 1X81 inverted microscope</td></tr><tr><td>Microscope camera</td><td>Photometrics</td><td>512B</td><td>Photometrics Cascadell 512B camera (4x UPlanFL N, 10x UPlanFL N, 20x UPlanFL N, 40x UPlanFL N objectives)</td></tr><tr><td>Microscope controller</td><td>Olympus</td><td>1X2-UCB</td><td>Olympus 1X2-UCB microscope controller</td></tr><tr><td>Microscope temperature controller</td><td>Solent Scientific</td><td>RS232</td><td>Maintained at 37&amp;deg;C</td></tr><tr><td>Penicillin/streptomycin</td><td>Sigma</td><td>A5955</td><td></td></tr><tr><td>Sodium Pyruvate</td><td>Sigma</td><td>S8636</td><td></td></tr><tr><td>Statistics software</td><td>Graphpad Prism</td><td>v7.0</td><td></td></tr><tr><td>STO feeder cells</td><td>ATCC</td><td>CRL-1503</td><td></td></tr><tr><td>Stereomicroscope</td><td>Nikon</td><td>SMZ</td><td></td></tr><tr><td>XY microscope stage controller</td><td>Applied Scientific Instrumentation (ASI)</td><td>MS-4400</td><td></td></tr></tbody></table>

dissection, culture and analysis of primary cranial neural crest cells from mouse for the study of neural crest cell delamination and migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

Using the procedure demonstrated here, mouse embryos were dissected from the uterus, and extraembryonic tissues were removed (Figure 1A&#8211;D). Embryos were somite staged (using only embryos at 5&#8211;8 somites (ss), Figure 1E,F). The cranial neural plate was then dissected and the neuroepithelium was isolated. Mesodermal cells, identified as loose, circular, mesenchymal cells, were gently brushed off (Figure 1G-L). The anterior neural plate can be explanted whole, in which case the neural crest tissue will emerge laterally and expand radially around the explant, or each neural plate border (right and left) can be explanted separately. This is particularly useful when explanting from genetic mutants.
Within 24 h, a region of premigratory (epithelial) cranial neural crest can clearly be seen surrounding the neural plate explant (Figure 2B). Furthermore, a subpopulation of neural crest cells have undergone epithelial to mesenchymal transition and appear fully mesenchymal (Figure 2). Thus, we have several concentric rings of distinct cells, with the neural plate (NP) in the center, the premigratory neural crest (pNC) in the intermediate circle, and a population of migratory neural crest (mNC) in the outside ring (Figure 2B). In order to trace NC cells, it is possible to use genetically modified mouse models as we show in Figure 2C. In this case, we have used the neural crest specific Wnt1::Cre;RosamTmG which results in NC cells being labeled in green. In these mice, cells express membrane tomato (mT, in red) unless they are expressing Cre recombinase. Recombination leads to cells expressing membrane green fluorescent protein (GFP, in green). The red cells shown at the center of the explant are neural plate cells. Some dorsal neural plate cells also express GFP; for long term culture, we would excise all of the cells in the center. For our purposes, the purity of the explant is sufficient to track the different neural crest cell populations. Where higher purity of the neural crest is necessary, this genetic labeling strategy can be combined with fluorescent activated cell sorting (FACS) to ensure purity of the population. Alternatively, it is possible to fix the explants and identify the NC population with antibody labeling.
It was also evident by 24 h that the characteristic concentric rings of premigratory and fully migratory NC cells of the explant cultures was not dependent nor governed by matrix choice (Figure 3). Explant cultures plated on both an ECM-based hydrogel and fibronectin formed comparable explant structures, comprising the three cell populations, NP, pNC and mNC (Figure 3A,C). Neural crest cell morphology was also comparable between those plated on the ECM-based hydrogel and fibronectin (Figure 3B,D). However, explants plated on fibronectin produced cells with more prominent lamellipodia at the cell leading edge, seemingly more polarized in the direction of migration (Figure 3B,D).
Once a population of migratory neural crest cells is evident, live cell imaging can be completed. Time-lapse microscopy is set to 10x magnification (18 h, 1 frame/5 min) for subsequent analysis of NC cell migration (Figure 4A). ImageJ Manual Tracking plug-in generates XY coordinates of individual cells over all frames of the time-lapse movies (Figure 4B). These coordinates can be processed using the migration software. This software enables visualization of individual cell tracks over time (Figure 4B) and can be used to quantify accumulated and Euclidean distance, as well as cell speed.
Time-lapse imaging data also provides a wealth of information on cell morphology during the migration of cranial neural crest cells (Figure 4C). By outlining individual cell membranes, cell area and perimeter measurements can be calculated from all frames of the movies (Figure 4C). These measurements allow for the subsequent quantification of cell area and circularity (Figure 4D). Figure 4C shows an analysis of cell shape changes over 18 h. Note that as cells migrate away from the explant, cell area significantly increases while cell circularity remains relatively constant (one-way ANOVA, Tukey&#8217;s multiple comparisons test) (Figure 4E,F). This suggests that as cells depart from the epithelial edge and lose cell-cell contacts, they show increased cell spread area. Cell circularity measures did not significantly change over time; however, short-term changes in circularity may be seen if an increased number of time points are quantified. Cell circularity measures can also provide interesting data on cell shape dynamics in the presence of a chemotactic cue or under confined conditions.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60051/60051fig1.jpg" /> 
Figure 1: Isolation of cranial neural crest explants from an e8.5 embryo. 
Images are stills from a video documenting the micro-dissection technique. (A&#8211;C) Dissection of the embryo from the uterus. (B&#8211;C) Using two sharp forceps, gently pull apart the muscular layer. Panel (D) shows embryo inside the visceral yolk sac (yellow line). Extract the embryo from the visceral yolk sac. (E) Lateral views of the embryo at stage 8.5 lateral. (F) Dorsal view of the embryo at stage 8.5. Count somites (ss) to determine the age of embryos; usually 5&#8211;8 ss (yellow circles in F). (G) Close up look at the cranial region of the embryo. Remove extraembryonic membranes from the cranial region; somites are marked with a yellow line. (H) Dissections of anterior neural plate are performed under the first branchial arch (yellow line). (I) Lateral view of anterior neural plate dissection. Neural folds, where neural crest cells arise, are marked with a yellow line. (J&#8211;L) Remove mesodermal tissue (fluffy mesenchymal cells) underlying anterior neural folds as much as possible before plating NP onto prepared culture dishes. Movie was taken using a stereo-microscope with a widefield apochromatic lens at 3.0X zoom (see the Table of Materials). <a href="https://www.jove.com/files/ftp_upload/60051/60051fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/60051/60051fig2.jpg" /> 
Figure 2: Murine cranial neural crest explant. 
(A) Schematic representation of the dorsal view of an e8.5 mouse embryo. The cranial region of the embryo is cut at the dashed line. The neural plate border (highlighted in red) is isolated from the surrounding mesoderm tissue and cultured for 24 h to allow the cranial neural crest to emigrate. Schematic adapted from22,23. (B) Left: Representative bright field image of a cranial neural crest explant 24 hours after plating. Three populations of cells are observed, which are also schematized on the right. NP = neural plate, pNC = pre-migratory neural crest and mNC = migratory neural crest. Scale bar = 250 &#956;m. (C) Higher magnification images of an explant from genetically labeled mouse (Wnt1::cre; RosamTmG). Cells without the Cre driver express membrane tomato (mT) in red. Expression of Cre under the control of a neural crest specific Wnt1 promoter leads to excision of the mT cassette and expression of membrane GFP (mG) in green. Nuclei are stained with Hoescht (in blue). Scale bar = 200 &#181;m. (D&#8211;D&#8217;&#8217;) Higher magnification images of migratory cells expressing membrane GFP (D). (D&#8217;) DNA is labeled with Hoescht (blue). (D&#8217;&#8217;) Merge of D and D&#8217;. Scale bar = 20 &#181;m. <a href="https://www.jove.com/files/ftp_upload/60051/60051fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/60051/60051fig3.jpg" /> 
Figure 3: Explants cultured on different substrates. 
(A&#8211;B) Phase contrast images of explants cultured on commercial ECM-hydrogel. (C&#8211;D) Explants cultured on 1 &#181;g/mL fibronectin. Neural plate (NP), premigratory (pNC) and migratory (mNC) neural crest cells can be distinguished by their differing cell morphologies. Scale bar = 100 &#956;m. <a href="https://www.jove.com/files/ftp_upload/60051/60051fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/60051/60051fig4.jpg" /> 
Figure 4: Quantification of cranial neural crest cell migration and cell shape dynamics. 
(A) Phase-contrast frames from time-lapse imaging of explant cultures overlaid with single neural crest cell tracks, using the ImageJ/Fiji Manual Cell Tracking plug-in. Ten representative mNC cells were manually tracked over 18 h (217 frames) and the XY coordinates were exported. Data are represented as an overlay dot and line plots. Cells were plated on 1 &#956;g/mL fibronectin. Scale bar = 200 &#956;m. (B) Representative trajectory plot of 10 mNC cells, generated using migration software. (C) Phase-contrast frames taken from time-lapse analysis of explant cultures. Dashed lines outline 8 representative migratory neural crest cells analyzed for cell shape dynamics when plated on 1 &#956;g/mL fibronectin. Scale bar = 200 &#956;m. (D) Schematic representation of the calculations used to quantify cell area and circularity. Cell morphology of the schematic is that of the cell highlighted in blue (C). Apx = pixel area, Npx = pixel number, A = area, P = perimeter (1 pixel = 1.60772 &#181;m2). (E&#8211;F) Quantification of cell area and cell circularity measures over time. Data represents mean &#177; SEM. Each dot represents one cell (n = 60), taken from&#160;3 independent experiments, and analyzed at 0 h, 6 h, 12 h and 18 h (ns non-significant, * p&#60;0.05, **** p &#60; 0.0001 one-way ANOVA, Tukey&#8217;s multiple comparisons test). <a href="https://www.jove.com/files/ftp_upload/60051/60051fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration at JoVE.com

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

dissection-culture-analysis-primary-cranial-neural-crest-cells-from

Research

JoVE Journal

Developmental Biology

10.7K Views.  King's College London. Dit goed gedefinieerde protocol kijkt naar primaire neurale kammigratie bij zoogdieren. We gebruiken primaire neurale kammigratie om geboorteafwijkingen te bestuderen die de neurale kam beïnvloeden. Deze aanpak stelt ons in staat om de neurale kuifcellen nader te bekijken als ze uit de neurale plaat tevoorschijn komen.Testen op celgedrag in weefsels die gemuteerde genen allures of na farmaceutische behandeling, maakt gezondheidsonderzoek voor de effecten van medicamenteuze behandeling...

Video: Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

Biology

Analysis of Trunk Neural Crest Cell Migration using a Modified Zigmond Chamber Assay

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after undergoing an epithelial-mesenchymal transition 1,2. Following EMT, NCCs migrate large distances along stereotypic pathways until they reach their targets. NCCs differentiate into a vast array of cell types including neurons, glia, melanocytes, and chromaffin cells 1-3. The ability of NCCs to reach and recognize their proper target locations is foundational for the appropriate formation of all structures containing trunk NCC-derived components 3. Elucidating the mechanisms of guidance for trunk NCC migration has therefore been a matter of great significance. Numerous molecules have been demonstrated to guide NCC migration 4. For instance, trunk NCCs are known to be repelled by negative guidance cues such as Semaphorin, Ephrin, and Slit ligands 5-8. However, not until recently have any chemoattractants of trunk NCCs been identified 9. 
Conventional in vitro approaches to studying the chemotactic behavior of adherent cells work best with immortalized, homogenously distributed cells, but are more challenging to apply to certain primary stem cell cultures that initially lack a homogenous distribution and rapidly differentiate (such as NCCs). One approach to homogenize the distribution of trunk NCCs for chemotaxis studies is to isolate trunk NCCs from primary NT explant cultures, then lift and replate them to be almost 100% confluent. However, this plating approach requires substantial amounts of time and effort to explant enough cells, is harsh, and distributes trunk NCCs in a dissimilar manner to that found in in vivo conditions. 
Here, we report an in vitro approach that is able to evaluate chemotaxis and other migratory responses of trunk NCCs without requiring a homogenous cell distribution. This technique utilizes time-lapse imaging of primary, unperturbed trunk NCCs inside a modified Zigmond chamber (a standard Zigmond chamber is described elsewhere10). By exposing trunk NCCs at the periphery of the culture to a chemotactant gradient that is perpendicular to their predicted natural directionality, alterations in migratory polarity induced by the applied chemotactant gradient can be detected. This technique is inexpensive, requires the culturing of only two NT explants per replicate treatment, avoids harsh cell lifting (such as trypsinization), leaves trunk NCCs in a more similar distribution to in vivo conditions, cuts down the amount of time between explantation and experimentation (which likely reduces the risk of differentiation), and allows time-lapse evaluation of numerous migratory characteristics.

An approach to analyze the migration of explanted cells (trunk neural crest cells) is described. This method is inexpensive, gentle, and capable of distinguishing chemotaxis from both chemokinesis and other influences on migratory polarity such as those derived from cell-cell interactions within the primary trunk neural crest cell culture.

Neural crest cells (NCCs) are a transient population of cells present in vertebrate development that emigrate from the dorsal neural tube (NT) after ...

Neuroscience

Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. Chimeric avian embryos, in which quail donor tissue is transplanted into a chick embryo in ovo, combine the power of indelible genetic labeling of cell populations with the ease of manipulation presented by the avian embryo.
Quail-chick chimeras are a classical tool for tracing migratory neural crest cells (NCCs)1-3. NCCs are a transient migratory population of cells in the embryo, which originate in the dorsal region of the developing neural tube4. They undergo an epithelial to mesenchymal transition and subsequently migrate to other regions of the embryo, where they differentiate into various cell types including cartilage5-13, melanocytes11,14-20, neurons and glia21-32. NCCs are multipotent, and their ultimate fate is influenced by 1) the region of the neural tube in which they originate along the rostro-caudal axis of the embryo11,33-37, 2) signals from neighboring cells as they migrate38-44, and 3) the microenvironment of their ultimate destination within the embryo45,46. Tracing these cells from their point of origin at the neural tube, to their final position and fate within the embryo, provides important insight into the developmental processes that regulate patterning and organogenesis.
Transplantation of complementary regions of donor neural tube (homotopic grafting) or different regions of donor neural tube (heterotopic grafting) can reveal differences in pre-specification of NCCs along the rostro-caudal axis2,47. This technique can be further adapted to transplant a unilateral compartment of the neural tube, such that one side is derived from donor tissue, and the contralateral side remains unperturbed in the host embryo, yielding an internal control within the same sample2,47. It can also be adapted for transplantation of brain segments in later embryos, after HH10, when the anterior neural tube has closed47.
Here we report techniques for generating quail-chick chimeras via neural tube transplantation, which allow for tracing of migratory NCCs derived from a discrete segment of the neural tube. Species-specific labeling of the donor-derived cells with the quail-specific QCPN antibody48-56 allows the researcher to distinguish donor and host cells at the experimental end point. This technique is straightforward, inexpensive, and has many applications, including fate-mapping, cell lineage tracing, and identifying pre-patterning events along the rostro-caudal axis45. Because of the ease of access to the avian embryo, the quail-chick graft technique may be combined with other manipulations, including but not limited to lens ablation40, injection of inhibitory molecules57,58, or genetic manipulation via electroporation of expression plasmids59-61, to identify the response of particular migratory streams of NCCs to perturbations in the embryo's developmental program. Furthermore, this grafting technique may also be used to generate other interspecific chimeric embryos such as quail-duck chimeras to study NCC contribution to craniofacial morphogenesis, or mouse-chick chimeras to combine the power of mouse genetics with the ease of manipulation of the avian embryo.62

An approach for analyzing migration and eventual fate of avian neural crest cells in quail-chick chimeric embryos is described. This method is a simple and straightforward technique for tracing neural crest cells during migration and differentiation that are otherwise difficult to distinguish within an unmanipulated chick embryo.

Avian embryos provide a unique platform for studying many vertebrate developmental processes, due to the easy access of the embryos within the egg. ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube is an essential process for NCC production and their subsequent migration toward target sites. The migratory route of NCCs, including the surrounding neural tube tissues, involves hyaluronan (HA)-rich extracellular matrix. To model NCC migration into these HA-rich surrounding tissues from the neural tube, a mixed substrate migration assay consisting of HA (average molecular weight: 1,200-1,400 kDa) and collagen type I (Col1) was established in this study. This migration assay demonstrates that NCC cell line, O9-1, cells are highly migratory on the mixed substrate and that the HA coating is degraded at the site of focal adhesions in the course of migration. This in vitro model can be useful for further exploration of the mechanistic basis involved in NCC migration. This protocol is also applicable for evaluating different substrates as scaffolds to study NCC migration.

In Vitro Investigation of the Effects of the Hyaluronan-Rich Extracellular Matrix on Neural Crest Cell Migration

This protocol outlines an in vitro migration experiment suitable for the functional analysis of the molecules involved in the in vivo migration of neural crest cells into the hyaluronan-rich extracellular matrix.

Neural crest cells (NCCs) are highly migratory cells that originate from the dorsal region of the neural tube. The emigration of NCCs from the neural tube ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

Cranial Neural Crest Cells Three-Dimensional In Vitro Differentiation Protocol for Multiplexed Assay

With their remarkable capacity to generate both ectodermal and mesenchymal derivatives, cranial neural crest cells (CNCC) have attracted a lot of interest in studying the mechanisms regulating cell fate decisions and plasticity. Originating in the dorsal neuroepithelium, this cell population is transient and relatively rare in the developing embryo - making functional tests, genomic screens, and biochemistry assays challenging to perform in vivo. To overcome these limitations, several methods have been developed to model CNCC development in vitro. Neurosphere (NS) based culturing methods provide a complex microenvironment that recapitulates the developing anterior neuroepithelium in 3D. These systems allow the growth of many NS in the same plate to generate a large amount of CNCC, but the produced NS present a high variability in shape, size, and number of CNCC formed - making quantitative assays difficult to perform. This protocol outlines a reproducible method for generating NS from mouse embryonic stem cells (mESC) in a 96-well format. NS generated in 96-well plates produce cranial neural crest cells (CNCC), which can be further cultured. By controlling the number of starting cells, this approach reduces variability in the size and shape between NS and increases reproducibility across experiments. Finally, this culture system is adaptable to several applications and offers a higher degree of flexibility, making it highly customizable and suitable for multiplexing experimental conditions.

We present a three-dimensional (3D) in vitro differentiation protocol generating neurospheres of reproducible size to produce cranial neural crest cells from mouse embryonic stem cells. We show that this methodology reduces variability compared to previous protocols and how it can be used for multiplexed assay to study cranial neural crest cell development.

With their remarkable capacity to generate both ectodermal and mesenchymal derivatives, cranial neural crest cells (CNCC) have attracted a lot of interest ...

Isolating Cranial Neural Folds From a Chick Embryo for a Neural Crest Cell Culture

Source: Jacques-Fricke, B. T., et al. <a href="https://app.jove.com/v/63799">Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures.</a> J. Vis. Exp. (2022).
This video illustrates a technique for isolating cranial neural folds from the midbrain region of chick embryos. It presents a detailed dissection process of the neural folds, their placement, and attachment on fibronectin-coated coverslips, followed by the observation of neural crest cell migration from the neural folds.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video