We thank Corinne A. Fairchild and Katie L. Vermillion, who participated in developing our version of the chick cranial neural fold culture protocol.

Any variety of Gallus gallus breeds may be used, including White Leghorn, Golden Sex Link, or Rhode Island Red. The chicken eggs used in the present study were of various breeds and obtained from multiple sources, including local farms and hatcheries.
1. Preparation of solutions and materials
<ol>
	<li>Prepare Ringer&#39;s solution by mixing 123.3 mM NaCl, 1.53 mM CaCl2, 4.96 mM KCl, 0.809 mM Na2HPO4, and 0.147 mM KH2PO4 (see Table of Materials). Adjust pH to 7.4 and filter sterilize into 100 mL bottles. Store in a clean, dry location. Do not wash bottles with soap (treat as tissue culture glassware).</li>
	<li>Prepare 1 mg/mL stock solution fibronectin (FN) (see Table of Materials) by dissolving FN in sterile Ringer&#39;s solution and store in 100 &#181;L aliquots at -80 &#176;C (stable for at least 4 years).</li>
	<li>Prepare complete culture media by supplementing L15 media with 0.8% L-glutamine, 0.08% penicillin/streptomycin, 10% FBS, and 10% chick embryo extract12. Create 3 mL aliquots and store at -20 &#176;C or -80 &#176;C. 
	NOTE: If cultures are incubated in a CO2 incubator, DMEM/F12 needs to be substituted for L15, which is buffered for air.</li>
	<li>Prepare 4% paraformaldehyde solution in 1x PBS. Adjust pH to 7.4. Create 10 mL aliquots and store them at -20 &#176;C. 
	CAUTION: Paraformaldehyde must be handled in a fume hood.</li>
	<li>Prepare filter paper support frames (approximately 1 cm x 1.5 cm rectangles of paper with two or three punched holes overlapping on the long axis).
	<ol>
		<li>Punch holes along the edge of a large piece of filter paper using a standard three-hole punch, cut/trim the punched edge into a strip, and then cut the strip between the holes into rectangles ~ 1.5 cm in length. Autoclave filter papers before use (optional).</li>
	</ol>
	</li>
	<li>Gather dissection tools, including fine forceps (#5), blunt forceps, dissection scissors, spring scissors, and sharpened tungsten needles16.</li>
</ol>
2. Embryo incubation
<ol>
	<li>Obtain fertilized eggs from any of the sources mentioned above.</li>
	<li>Place the fertilized eggs in an upright position (long axis of the egg vertical with the pointed end down/blunt side up) inside a humidified incubator at 38 &#176;C (100 &#176;F).</li>
	<li>Incubate until four to seven somites have formed (stages 8+-9)17, which takes approximately 35 h. 
	NOTE: Incubation time can vary significantly depending on the strain, age of hens, season, etc., and must be experimentally determined. Programmable outlet timers are useful for initiating incubation during the night to obtain desired timing.</li>
	<li>After removing from the incubator, thoroughly spray the eggs with 70% ethanol and let them dry to disinfect the shells. 
	&#8203;NOTE: Embryos can be collected immediately, but letting eggs cool to room temperature decreases the fragility of the yolks and embryo loss during collection.</li>
</ol>
3. Preparation of culture dishes
<ol>
	<li>Select neural fold culture dishes (see Table of Materials) appropriate for the downstream application.
	<ol>
		<li>For cultures that will be fixed and stained, use glass coverslips placed into multi-well tissue culture dishes. 
		NOTE: Coverslips that match the well size will move less with fluid mixing, making explants less likely to shift and more likely to adhere to the coverslip rather than the dish bottom.</li>
		<li>Select glass-bottom single chamber or divided chamber dishes (see Table of Materials) for live imaging with an inverted microscope.</li>
		<li>For live imaging on an upright or stereo microscope, use 6-, 12-, or 24-well optically appropriate tissue culture plates.</li>
		<li>Select plastic tissue culture dishes for the mass collection of migratory neural crest cells for high-throughput analyses.</li>
	</ol>
	</li>
	<li>Add 10 U/mL of penicillin and 10 &#181;g/mL of streptomycin to a 100 mL bottle of sterile Ringer&#39;s solution (for example, 100 &#181;L of 10,000 U/mL penicillin and 10,000 &#181;g/mL streptomycin, to make Ringer&#39;s P/S). Use Ringer&#39;s P/S within 1 week.</li>
	<li>Thaw an aliquot of 1 mg/mL of FN on ice. Dilute with Ringer&#39;s P/S to a concentration of 10-100 &#181;g/mL of FN.</li>
	<li>Pipet enough FN solution to cover the bottom of the well or dish. For example, 100 &#181;L per well in a 24-well plate, 500 &#181;L for a 35 mm dish.</li>
	<li>Replace the lid and incubate dishes or plates with FN solution in a humidified incubator (or a covered tray with distilled water-soaked paper towels) at 38 &#176;C (100 &#176;F) for at least 1 h while dissecting neural folds.</li>
</ol>
4. Isolation of chick embryos
<ol>
	<li>Take care to maintain the orientation of the egg (the embryo floats to the top of the yolk during incubation). Use scissors or blunt forceps to poke a small hole in the shell, about 1/4-1/3 of the way down the length of the egg.
	<ol>
		<li>Carefully insert the tip of scissors or blunt forceps into the small hole, ensuring not to disrupt the yolk, and cut through the eggshell around the egg, removing the top of the eggshell (Figure 2A).</li>
	</ol>
	</li>
	<li>Position the embryo for isolation. 
	NOTE: If the embryo is not ideally located in the shell cup, use blunt forceps to carefully turn the yolk, so the embryo is on the top. Alternatively, pour the yolk into a gloved, cupped hand, being careful not to break the yolk and allowing the albumin to drain away (Figure 2B). Then, the embryo can be positioned by moving the yolk from hand to hand.</li>
	<li>Prepare the embryo by gently using the flat edge of closed blunt forceps to wipe away any excess albumin that remains on the yolk&#39;s surface covering the embryo (Figure 2C). Excess albumin can also be removed using gentle pressure with a delicate task wiper. 
	NOTE: Once albumin is cleared away, the yolk&#39;s surface should appear textured instead of smooth. Failure to wipe away sufficient albumin will inhibit the adhesion of the filter paper support in section 4, step 4.</li>
	<li>Use forceps to place a filter paper support frame over the embryo, with the embryo in the frame&#39;s window. Gently press down on the filter paper to adhere it onto the yolk.</li>
	<li>Cut around the outside of the filter paper frame with dissection scissors (Figure 2D). Use forceps or scissor tips to grasp the edge of the frame and gently lift the embryo away from the yolk (Figure 2E). Lifting away at an angle will help remove the embryo cleanly from the yolk.</li>
	<li>Place the embryo with the paper frame side down (embryo ventral side up) into a 60 mm or 100 mm Petri dish filled with Ringer&#39;s P/S (Figure 2F). Keep the dish of embryos on ice if collecting for an RNA- or protein-sensitive downstream application. 
	&#8203;NOTE: Failure to place embryos ventral side up risks detaching them from their paper frames. Multiple embryos can be collected and stored in Ringer&#39;s P/S for up to 1 h before moving to the neural fold dissection steps.</li>
</ol>
5. Dissecting neural folds
<ol>
	<li>Transfer an embryo to a clean dish containing Ringer&#39;s P/S solution and rinse the embryo by holding the filter paper frame with forceps and gently swishing back and forth to clear away any yolk that obscures the view of the embryo. Exchange the Ringer&#39;s P/S or transfer to a fresh dish if it becomes cloudy.</li>
	<li>Position the embryo dorsal/frame side up under a dissecting microscope. Leaving the embryo on the paper frame to keep it stretched taut and held in place, remove the vitelline membrane using forceps to expose the neural folds. 
	NOTE: If the embryo falls off the paper frame, it can be laid out flat in the dish or pinned to a sylgard coated dish18 for dissection.</li>
	<li>Using spring scissors or a sharpened tungsten needle, carefully excise the midbrain neural folds. Include tissue caudal to the expanding optic vesicles and rostral to the hindbrain, where rhombomere constrictions are just beginning to appear (the cardiac crescent is also a useful indicator, Figure 3B,C). Take care to excise the dorsal-most aspect of the neural fold with minimal contaminating neural tube and non-neural ectoderm (Figure 3C).</li>
	<li>Transfer the neural folds to a clean dish containing Ringer&#39;s P/S using a P20 pipettor or a sterile, glass Pasteur pipette rinsed with yolky Ringer&#39;s P/S (this blocks the plastic or glass to prevent the tissue from sticking). Store collected folds on ice while dissecting additional folds.</li>
</ol>
6. Plating neural folds
<ol>
	<li>Thaw an aliquot of complete culture media (section 1, step 3). Add 100 U/mL of penicillin and 100 &#181;g/mL of streptomycin and filter sterilize. Keep prepared media at 37-38 &#176;C while performing other steps.</li>
	<li>Remove culture dishes from the incubator (section 3, step 5). Using a pipettor or Pasteur pipette, remove the FN solution from coverslips, dishes, or wells. After rinsing the FN-coated substrate with Ringer&#39;s P/S, add an appropriate volume of complete culture media to the dishes or wells (500 &#181;L for wells in a 24-well plate, 2 mL for a 35 mm dish or a six-well plate). 
	NOTE: Smaller volumes can preserve expensive reagents (e.g., as low as 200 &#181;L for a 24-well plate) but ensure that the dish is sufficiently humidified to avoid evaporation.</li>
	<li>Using a p20 or p200 pipettor, first, rinse the pipette tip with yolky Ringer&#39;s P/S to block the plastic and prevent the tissue from sticking. Then, transfer the isolated neural folds onto the FN coated coverslips, taking care to transfer as little Ringer&#39;s P/S as possible. Place the fold(s) toward the center of the FN-coated coverslip. 
	NOTE: One or a few neural folds can be plated on a 12 mm coverslip in a 19 mm well, up to 50 on a 35 mm plate.</li>
	<li>After allowing the explants to settle for 10-15 min, place them into a humidified chamber at 38 &#176;C (100 &#176;F) by slowly and carefully carrying the culture dishes with plated neural folds. This will minimize the shifting of neural folds in the wells, ensuring they remain dispersed and adhere to any coverslips. 
	NOTE: When using L15 (not DMEM), explant culture dishes can also be incubated in an egg incubator using a covered tray with moistened paper towels.</li>
	<li>Incubate neural fold cultures in the humidified incubator for the duration of active migration (about 16-20 h total, Figure 4).</li>
</ol>
7. Fixing and staining of cultured migratory NCCs for morphological analysis
<ol>
	<li>Remove the culture media with a Pasteur pipette and rinse the wells with filter-sterilized 1x PBS.</li>
	<li>Add 4% paraformaldehyde for 15 min at room temperature. 
	NOTE: Fixation time may need to be experimentally determined. It is also possible to add paraformaldehyde directly to media for 10 min (50:50), remove, and replace with undiluted 4% paraformaldehyde for 10 min to retain more delicate cellular structures.</li>
	<li>Remove paraformaldehyde and rinse three times with 1x PBS.</li>
	<li>Stain fixed NCCs with an appropriate dye for morphological assessment. As an example, staining with Oregon Green conjugated phalloidin is detailed here. 
	NOTE: Visualizing the actin cytoskeleton with phalloidin19 reveals the structural complexity of migratory NCC with relatively uniform staining intensity; however, phalloidin may not highlight all cell areas. Dyes that label the plasma membrane (e.g., wheat germ agglutinin or DiI) or cytoplasm can also be used, but complicate the imaging of fine protrusions relative to the brightly staining cell body. In addition, immunofluorescence can be performed concurrently to label neural crest cells with HNK-1 or to determine the subcellular localization of a protein of interest7,20.
	<ol>
		<li>Remove the final PBS rinse and add PBS + 0.5% Triton X-100 (PBST) + 5% serum (FBS or from another animal appropriate for costaining with an antibody) to cover the coverslips and incubate for 10 min on a platform shaker at room temperature.</li>
		<li>Pipet 200 nM of Oregon Green conjugated phalloidin (diluted in PBST + 5% serum) onto a smooth surface (such as flexible paraffin sealing film) for each coverslip. For a 12 mm coverslip, pipette a 30 &#181;L volume.</li>
		<li>Using a pair of forceps, remove the coverslip from the PBST + 5% serum, ensuring to maintain the orientation of the cells facing upward. Briefly touch the edge of the coverslip to a delicate task wiper to wick off excess liquid.</li>
		<li>Gently place each coverslip cell side down onto the drop of diluted phalloidin. Incubate for 30 min at room temperature. Keep the coverslips in the dark during staining (cover with an aluminum foil-covered dish or place in a drawer).</li>
		<li>During the incubation, add PBST to the wells of the culture dish (750 &#181;L of PBST for each well of a 24-well plate).</li>
		<li>After the incubation period, lift coverslips off of the staining solution and place them back into the culture dish, flipping the coverslip over so the cell side is up. Ensure the coverslip is covered with PBST and place on a platform shaker for 10 min, keeping the coverslips covered and in the dark. Remove PBST and repeat this step two times, for a total of three 10 min washes.</li>
		<li>Place one drop (per coverslip) of mounting media (see Table of Materials) onto a microscope slide. 25 &#181;L volume works well for a 12 mm coverslip.</li>
		<li>After the final wash with PBST, touch the edge of the coverslip to a delicate task wiper to remove excess liquid, keeping track of the cell side of the coverslip.</li>
		<li>Mount the coverslip cell side down by slowly lowering the coverslip at an angle onto the mounting media to avoid creating bubbles. Allow the media to set before imaging.</li>
	</ol>
	</li>
</ol>
8. Morphological assessment of cultured migratory NCCs
<ol>
	<li>Image stained cells and export as .tiff files. 
	NOTE: A 40x objective works well for imaging cultures, but objectives ranging from 10x (to capture a whole field of migratory NCCs) to 100x (single NCCs) can be used to collect images for morphological assessment. Images in the present study were captured using an inverted multimodal imaging platform (see Table of Materials).</li>
	<li>Upload the images into an image analysis software (ImageJ21, see Table of Materials). Click on Image &#62; Duplicate to create a second copy of each image to use for analysis, thus leaving the original unedited, as most image processing cannot be reversed.</li>
	<li>Click on Image &#62; Adjust &#62; Brightness/Contrast and use sliding bars to adjust the brightness or contrast of the images.</li>
	<li>Convert the images to grayscale following the steps below.
	<ol>
		<li>If images are exported in RGB, they will upload in RGB as a merged channel. To separate the images, click on Image &#62; Color &#62; Split Channels to acquire single-channel grayscale images.</li>
		<li>If images are already separated, go to Image &#62; Type &#62; 8-bit to convert file to grayscale.</li>
	</ol>
	</li>
	<li>Click on Image &#62; Adjust &#62; Threshold to convert to a binary image where pixels are identified as cells (foreground; black) or background (white). Use the sliding bar and/or click on Auto to select the Threshold setting that clearly defines cells from the background.
	<ol>
		<li>If cells are overlapping or staining is not continuous, it may be necessary to process further using the Watershed or Fill Holes functions22. This will separate cells from one another or fill in gaps within cells to be analyzed. Click on Process &#62; Binary &#62; Make Binary to first convert the image into an 8-bit binary format. Then click on Process &#62; Binary &#62; Watershed or Fill Holes. 
		NOTE: The software will make its best fit for where signals need to be separated or filled in, which can be adjusted further using plug-ins if necessary.</li>
	</ol>
	</li>
	<li>Select the measurements to be captured. Click on Analyze &#62; Set Measurements and check the Shape Descriptors boxes for analysis of Circularity (Circ.), Aspect Ratio (AR), Roundness (Round), and Solidity. 
	NOTE: Other measurements may also be included, like Area and Perimeter, depending on the type of analysis needed.</li>
	<li>Click on Analyze &#62; Analyze Particles, and set the parameters from the &#34;Analyze Particles&#34; menu.
	<ol>
		<li>Under &#34;Size&#34;, roughly estimate how large the cells or particles of interest are in pixels, as background signal or smaller cell debris will also be included if the Size parameter remains set at 0-Infinity (for example, set as 500-Infinity in Figure 5). 
		NOTE: Adjusting the Size to a tighter range can exclude smaller or larger particles than the ones needed for analysis.</li>
		<li>Under &#34;Circularity&#34;, leave at 0-1.0 to measure all cell shapes in the image.</li>
		<li>Under &#34;Show&#34;, select Bare Outlines from the drop-down menu to inspect the cell outlines selected with the &#34;Analyze Particles&#34; function. Scan the outlines to ensure that overlapping cells are distinguished, but cells are not divided unnecessarily. Also, check the menu boxes for &#34;Display Results&#34;, &#34;Summarize&#34;, and &#34;Add to Manager&#34;.</li>
	</ol>
	</li>
	<li>Once all the parameters have been set, click on Ok. 
	NOTE: Four separate windows will pop up, showing (1) the bare outlines of the cells identified in the image, (2) the cells counted in the image, (3) the results of each cell&#39;s measurements, and (4) a summary of the total number of cells counted and their averaged measurements.
	<ol>
		<li>If debris was counted, overlapping cells were counted as one, single cells were counted as multiples, or many cells were omitted from the count, go back to the original, unedited image, make another duplicate, and adjust &#34;Threshold&#34; or other parameters.</li>
	</ol>
	</li>
</ol>

<ol><li>Pla, P., Monsoro-Burq, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+neural+border%3A+Induction%2C+specification+and+maturation+of+the+territory+that+generates+neural+crest+cells%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">The neural border: Induction, specification and maturation of the territory that generates neural crest cells.</a> Developmental Biology. 444, 36-46 (2018).</li>
<li>Tang, W., Bronner, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+crest+lineage+analysis%3A+From+past+to+future+trajectory%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Neural crest lineage analysis: From past to future trajectory.</a> Development. 147 (20), (2021).</li>
<li>Piacentino, M. L., Li, Y., Bronner, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Epithelial-to-mesenchymal+transition+and+different+migration+strategies+as+viewed+from+the+neural+crest%5BTitle%5D)+AND+%22Current+Opinion+in+Cell+Biology%22%5BJournal%5D)">Epithelial-to-mesenchymal transition and different migration strategies as viewed from the neural crest.</a> Current Opinion in Cell Biology. 66, 43-50 (2020).</li>
<li>McLennan, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+crest+cells+bulldoze+through+the+microenvironment+using+Aquaporin+1+to+stabilize+filopodia%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">Neural crest cells bulldoze through the microenvironment using Aquaporin 1 to stabilize filopodia.</a> Development. 147 (1), 185231(2020).</li>
<li>Carmona-Fontaine, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Complement+fragment+C3a+controls+mutual+cell+attraction+during+collective+cell+migration%5BTitle%5D)+AND+%22Developmental+Cell%22%5BJournal%5D)">Complement fragment C3a controls mutual cell attraction during collective cell migration.</a> Developmental Cell. 21 (6), 1026-1037 (2011).</li>
<li>Giovannone, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Slits+affect+the+timely+migration+of+neural+crest+cells+via+robo+receptor%5BTitle%5D)+AND+%22Developmental+Dynamics%22%5BJournal%5D)">Slits affect the timely migration of neural crest cells via robo receptor.</a> Developmental Dynamics. 241 (8), 1274-1288 (2012).</li>
<li>Vermillion, K. L., Lidberg, K. A., Gammill, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cytoplasmic+protein+methylation+is+essential+for+neural+crest+migration%5BTitle%5D)+AND+%22Journal+of+Cell+Biology%22%5BJournal%5D)">Cytoplasmic protein methylation is essential for neural crest migration.</a> Journal of Cell Biology. 204 (1), 95-109 (2014).</li>
<li>Yang, X., Li, J., Zeng, W., Li, C., Mao, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Elongator+Protein+3+%28Elp3%29+stabilizes+Snail1+and+regulates+neural+crest+migration+in+Xenopus%5BTitle%5D)+AND+%22Scientific+Reports%22%5BJournal%5D)">Elongator Protein 3 (Elp3) stabilizes Snail1 and regulates neural crest migration in Xenopus.</a> Scientific Reports. 6 (1), 1-9 (2016).</li>
<li>Gonzalez Malagon, S. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Glycogen+synthase+kinase+3+controls+migration+of+the+neural+crest+lineage+in+mouse+and+Xenopus%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">Glycogen synthase kinase 3 controls migration of the neural crest lineage in mouse and Xenopus.</a> Nature Communications. 9 (1), 1-15 (2018).</li>
<li>Bhattacharya, D., Azambuja, A. P., Simoes-Costa, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Metabolic+reprogramming+promotes+neural+crest+migration+via+yap%2Ftead+signaling%5BTitle%5D)+AND+%22Developmental+Cell%22%5BJournal%5D)">Metabolic reprogramming promotes neural crest migration via yap/tead signaling.</a> Developmental Cell. 53 (2), 199-211 (2020).</li>
<li>Jacques-Fricke, B. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Profiling+NSD3-dependent+neural+crest+gene+expression+reveals+known+and+novel+candidate+regulatory+factors%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Profiling NSD3-dependent neural crest gene expression reveals known and novel candidate regulatory factors.</a> Developmental Biology. 475, 118-130 (2021).</li>
<li>Bronner-Fraser, M., García-Castro, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chapter+4+manipulations+of+neural+crest+cells+or+their+migratory+pathways%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Chapter 4 manipulations of neural crest cells or their migratory pathways.</a> Methods in Cell Biology. 87, 75-96 (2008).</li>
<li>Milet, C., Monsoro-Burq, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissection+of+xenopus+laevis+neural+crest+for+in+vitro+explant+culture+or+in+vivo+transplantation%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation.</a> Journal of Visualized Experiments. (85), e51118(2014).</li>
<li>Malagon, S. G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dissection%2C+culture+and+analysis+of+primary+cranial+neural+crest+cells+from+mouse+for+the+study+of+neural+crest+cell+delamination+and+migration%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">Dissection, culture and analysis of primary cranial neural crest cells from mouse for the study of neural crest cell delamination and migration.</a> Journal of Visualized Experiments. (152), e60051(2019).</li>
<li>Theveneau, E., Mayor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+crest+delamination+and+migration%3A+From+epithelium-to-mesenchyme+transition+to+collective+cell+migration%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Neural crest delamination and migration: From epithelium-to-mesenchyme transition to collective cell migration.</a> Developmental Biology. 366 (1), 34-54 (2012).</li>
<li>Conrad, G. W., Bee, J. A., Roche, S. M., Teillet, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fabrication+of+microscalpels+by+electrolysis+of+tungsten+wire+in+a+meniscus%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Fabrication of microscalpels by electrolysis of tungsten wire in a meniscus.</a> Journal of Neuroscience Methods. 50 (1), 123-127 (1993).</li>
<li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+series+of+normal+stages+in+the+development+of+the+chick+embryo%5BTitle%5D)+AND+%22Journal+of+Morphology%22%5BJournal%5D)">A series of normal stages in the development of the chick embryo.</a> Journal of Morphology. 88 (1), 49-92 (1951).</li>
<li>Gammill, L. S., Jacques-Fricke, B., Roffers-Agarwal, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Embryological+and+genetic+manipulation+of+chick+development%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Embryological and genetic manipulation of chick development.</a> Methods in Molecular Biology. 1920, 75-97 (2019).</li>
<li>Vandekerckhove, J., Deboben, A., Nassal, M., Wieland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+phalloidin+binding+site+of+F-actin%5BTitle%5D)+AND+%22The+EMBO+Journal%22%5BJournal%5D)">The phalloidin binding site of F-actin.</a> The EMBO Journal. 4 (11), 2815-2818 (1985).</li>
<li>Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+the+early+stages+of+trunk+neural+crest+migration+in+avian+embryos+using+monoclonal+antibody+HNK-1%5BTitle%5D)+AND+%22Developmental+Biology%22%5BJournal%5D)">Analysis of the early stages of trunk neural crest migration in avian embryos using monoclonal antibody HNK-1.</a> Developmental Biology. 115 (1), 44-55 (1986).</li>
<li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Fiji%3A+An+open-source+platform+for+biological-image+analysis%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Fiji: An open-source platform for biological-image analysis.</a> Nature Methods. 9 (7), 676-682 (2012).</li>
<li>Soille, P., Vincent, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Determining+watersheds+in+digital+pictures+via+flooding+simulations%5BTitle%5D)+AND+%22Visual+Communications+and+Image+Processing+%2790%3A+Fifth+in+a+Series%22%5BJournal%5D)">Determining watersheds in digital pictures via flooding simulations.</a> Visual Communications and Image Processing '90: Fifth in a Series. (1360), 240-250 (1990).</li>
<li>Haupt, A., Minc, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((How+cells+sense+their+own+shape+-+mechanisms+to+probe+cell+geometry+and+their+implications+in+cellular+organization+and+function%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">How cells sense their own shape - mechanisms to probe cell geometry and their implications in cellular organization and function.</a> Journal of Cell Science. 131 (6), (2018).</li>
<li>Ezin, M., Fraser, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chapter+11+time-lapse+imaging+of+the+early+avian+embryo%5BTitle%5D)+AND+%22Methods+in+Cell+Biology%22%5BJournal%5D)">Chapter 11 time-lapse imaging of the early avian embryo.</a> Methods in Cell Biology. 87, 211-236 (2008).</li>
<li>Kulesa, P. M., Bailey, C. M., Cooper, C., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+ovo+live+imaging+of+avian+embryos%5BTitle%5D)+AND+%22Cold+Spring+Harbor+Protocols%22%5BJournal%5D)">In ovo live imaging of avian embryos.</a> Cold Spring Harbor Protocols. 5 (6), (2010).</li>
<li>McKinney, M. C., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Live+imaging+of+the+neural+crest+cell+epithelial-to-mesenchymal+transition+in+the+chick+embryo%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Live imaging of the neural crest cell epithelial-to-mesenchymal transition in the chick embryo.</a> Methods in Molecular Biology. 2179, 107-114 (2021).</li>
<li>Gustafson, C. M., Roffers-Agarwal, J., Gammill, L. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chick+cranial+neural+crest+cells+release+extracellular+vesicles+that+are+critical+for+their+migration%5BTitle%5D)+AND+%22Journal+of+Cell+Science%22%5BJournal%5D)">Chick cranial neural crest cells release extracellular vesicles that are critical for their migration.</a> Journal of Cell Science. , (2022).</li>
<li>Williams, R., Sauka-Spengler, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+ovo+electroporation+of+early+chicken+embryos%5BTitle%5D)+AND+%22STAR+Protocols%22%5BJournal%5D)">Ex ovo electroporation of early chicken embryos.</a> STAR Protocols. 2 (2), 100424(2021).</li>
<li>Moulton, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+morpholinos+to+control+gene+expression%5BTitle%5D)+AND+%22Current+Protocols+in+Nucleic+Acid+Chemistry%22%5BJournal%5D)">Using morpholinos to control gene expression.</a> Current Protocols in Nucleic Acid Chemistry. 68 (1), 4-30 (2017).</li>
<li>Gandhi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+single-plasmid+approach+for+genome+editing+coupled+with+long-term+lineage+analysis+in+chick+embryos%5BTitle%5D)+AND+%22Development%22%5BJournal%5D)">A single-plasmid approach for genome editing coupled with long-term lineage analysis in chick embryos.</a> Development. 148 (7), (2021).</li>
<li>Williams, R. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Reconstruction+of+the+Global+Neural+Crest+Gene+Regulatory+Network+In+Vivo%5BTitle%5D)+AND+%22Developmental+Cell%22%5BJournal%5D)">Reconstruction of the Global Neural Crest Gene Regulatory Network In Vivo.</a> Developmental Cell. 51 (2), 255-267 (2019).</li>
</ol>

The authors have no conflicts of interest.

The technique described here provides an adaptable method of isolating chick neural folds and plating them to create cultures of migratory cranial NCCs. These cultures provide simplified 2D conditions for easy analysis of chick NCC migration and morphology that can supplement more technically challenging in ovo imaging methods24,25,26. While this in vitro method is relatively simple, consistent results depend o...

Neural crest cells (NCCs) are a transient cell population in vertebrate embryos. NCCs are specified at the borders of the neural plate and undergo an epithelial-to-mesenchymal transition (EMT) to migrate from the dorsal neural tube1. After EMT, NCCs disperse extensively throughout the embryo, ultimately differentiating and contributing to various structures, including the craniofacial skeleton, outflow tract of the heart, and the majority of the peripheral nervous system2. Changes in cell polarity, the cytoskeleton, and adhesion properties underly this shift from a premigratory to a migratory cell population3. Studying NCC EMT and migration provides insights into fundamental mechanisms of cell motility and informs efforts to prevent and treat birth defects and cancer metastasis.
While in vivo analysis is vital for understanding NCC developmental processes in an embryonic context, in vitro methods offer visual and physical accessibility that facilitate additional experimental avenues. In a simplified 2D environment, NCC morphology, cytoskeletal structures, and distance migrated can be evaluated. Moreover, the effects of genetic or soluble factor perturbation on migratory behaviors of motile NCCs can be analyzed4,5,6,7,8,9,10. In addition, isolated premigratory or migratory NCCs can be collected, pooled, and used for high-throughput methodologies to study the developmental regulation of NCCs through proteomic, transcriptomic, and epigenomic profiling7,11. While methods are available for preparing cranial NCCs from various developmental model organisms12,13,14, this article demonstrates the mechanics of the approach for those first learning to culture cranial NCC from chick embryos.
The current protocol describes a versatile technique for preparing chick cranial NCC cultures (Figure 1). Because NCCs migrate readily from explanted neural folds onto a culture substrate, chick NCCs naturally segregate from embryonic tissue, and primary cultures are easily generated. As midbrain NCCs migrate&#160;en masse&#160;from the cranial neural folds (in contrast to the protracted, cell-by-cell delamination in the trunk15), these cultures consist mainly of migratory cranial neural crest cells, with initial neural fold excision providing a collection method for premigratory NCCs.&#160;A basic method for dissecting and culturing chick cranial neural folds is detailed, and suggestions for different applications and variations on this method are offered.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63799/63799fig01v2.jpg" /> 
Figure 1: Schematic overview of the chick cranial neural fold culture protocol. (A,B) Cranial neural folds (outlined in blue) are excised from a chick embryo with five somites (shown in dorsal view in A). Grey bands, cardiac crescent. (C) When plated on fibronectin, migratory neural crest cells emerge from the neural folds and disperse onto the substrate. <a href="https://www.jove.com/files/ftp_upload/63799/63799fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>AxioObserver equipped with an LSM710 confocal scan head controlled by ZEN 3.0 SR software&amp;nbsp;</td><td>Zeiss</td><td></td><td>Used alpha Plan-Apochromat 100x/1.46 Oil DIC M27 objective</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C3306</td><td></td></tr><tr><td>Chamber dishes (glass bottom, single or divided)</td><td>MatTek; Cell Vis</td><td>P35G-1.5-14-C (MatTek) X000NOJQGX (Cellvis)&lt;br/&gt; X000NOK1OJ (Cellvis)</td><td>Single chamber 35 mm or 4 chamber 35 mm</td></tr><tr><td>Cover glass</td><td>Carolina Biological Supply Company</td><td>633029, 633031, 633033, 633035, 633037</td><td>circles, 0.13&amp;ndash;0.17 mm thickness, available in 12-25 mm diameter&amp;nbsp;</td></tr><tr><td>DMEM/F12</td><td>ThermoFisher Scientific</td><td>11320033</td><td>Alternative for L15 media</td></tr><tr><td>Egg incubator</td><td>Sportsman</td><td>1502</td><td></td></tr><tr><td>FBS&amp;nbsp;</td><td>Life Technologies</td><td>10437-028</td><td></td></tr><tr><td>Fibronectin</td><td>Fisher Scientific</td><td>CB-40008A</td><td></td></tr><tr><td>Filter paper</td><td>Whatman</td><td></td><td>grade 3MM chromatography</td></tr><tr><td>Forceps (blunt)</td><td>Fisher Scientific; Thomas Scientific</td><td>08-890 (Fisher);1141W97 (Thomas)</td><td></td></tr><tr><td>Forceps (fine)</td><td>Fine Science Tools</td><td>11252-20</td><td>Dumont #5</td></tr><tr><td>Image J</td><td>https://fiji.sc/</td><td></td><td>Free image analysis software</td></tr><tr><td>KCl</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>P0662</td><td></td></tr><tr><td>L15 media</td><td>Invitrogen</td><td>11415064</td><td></td></tr><tr><td>L-glutamine</td><td>Invitrogen</td><td>25030</td><td></td></tr><tr><td>Mounting Media (Vectashield or ProLong Gold)</td><td>Vector Laboratories; Thermofisher Scientific</td><td>H-1700 (Vectashield); P36930 (ProLong Gold)</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>S9638</td><td></td></tr><tr><td>NaCl</td><td>Sigma-Aldrich</td><td>S9888</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>P6148</td><td></td></tr><tr><td>Penicillin/streptomycin</td><td>Life Technologies</td><td>15140-148</td><td>10,000 Units/mL Penicillin; 10,000 mg/mL Streptomycin</td></tr><tr><td>Petri Dishes</td><td>VWR (or similar)</td><td></td><td>60 mm, 100 mm</td></tr><tr><td>Phalloidin</td><td>Sigma-Aldrich</td><td>P1951</td><td>multiple flurophores available</td></tr><tr><td>Pin holder</td><td>Fine Science Tools</td><td>26016-12</td><td>For tungsten needle (alternative for spring scissors)</td></tr><tr><td>Scissors (dissection)</td><td>Fine Science Tools</td><td>14061-10</td><td></td></tr><tr><td>Spring Scissors</td><td>Fine Science Tools</td><td>15000-08</td><td>2.5 mm cutting edge (alternative for tungsten needle)</td></tr><tr><td>Sylgard</td><td>Krayden</td><td>Sylgard 184</td><td></td></tr><tr><td>Syringe Filters</td><td>Sigma-Aldrich</td><td>SLGVM33RS</td><td>Millex-GV Syringe Filter Unit, 0.22 &amp;micro;m, PVDF, 33 mm, gamma sterilized</td></tr><tr><td>Tissue culture dishes</td><td>Sarstedt</td><td>83-3900</td><td>35 mm culture dishes for bulk neural fold cultures</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100</td><td></td></tr><tr><td>Tungsten wire</td><td>Variety of sources</td><td></td><td>0.01&quot; diameter for tungsten needle (alternative for spring scissors)</td></tr></tbody></table>

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.

An overview of the present protocol is shown in Figure 1. The incubated eggs were opened, and the yolk, with the embryo on the surface, was isolated by gently pouring into the palm of a gloved hand (Figure 2A,B). After clearing away the albumin (Figure 2C), filter paper frames were applied to the yolk membrane surrounding the embryo to facilitate cutting and lifting the embryo from the yolk, which begins to spill aw...

Watch this Scientific Journal Video about Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures at JoVE.com

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

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 ...

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Research

JoVE Journal

Developmental Biology

2.4K Views.  Hamline University. Neurale kamcellen emigreren uit schedelneuraalplooien in embryo's of wanneer ze in cultuur worden geplaatst. Dit biedt een handige methode om primaire migrerende neurale crest-cellen te isoleren zonder celdissociatie. Terwijl neurale crestmigratie meestal plaatsvindt binnen de driedimensionale embryonale omgeving, maakt tweedimensionale cultuur visualisatie en onderzoek van migratiegerelateerde processen mogelijk.Hoewel er craniale neurale vouwcultuurprotocollen bestaan, vereist deze m...

Video: Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Production of Chick Embryo Extract for the Cultivation of Murine Neural Crest Stem Cells

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent progenitor cells to be an integral part of the neural crest1. Phenotypically, neural crest stem cells (NCSC) are defined by simultaneously expressing p75 (low-affine nerve growth factor receptor, LNGFR) and SOX10 during their migration from the neural crest2,3,4,5. These progenitor cells can differentiate into smooth muscle cells, chromaffin cells, neurons and glial cells, as well as melanocytes, cartilage and bone6,7,8,9. To cultivate NCSC in vitro, a special neural crest stem cell medium (NCSCM) is required10. The most complex part of the NCSCM is the preparation of chick embryo extract (CEE) representing an essential source of growth factors for the NCSC as well as for other types of neural explants. Other NCSCM ingredients beside CEE are commercially available. Producing CCE using laboratory standard equipment it is of high importance to know about the challenging details as the isolation, maceration, centrifugation, and filtration processes. In this protocol we describe accurate techniques to produce a maximized amount of pure and high quality CEE.

To cultivate neural crest stem cells (NCSC) in vitro, a special medium (NCSCM) is required. Essential part of NCSCM is chick embryo extract (CEE). We here describe accurate techniques to produce a maximized amount of pure and high quality CEE, including details as the isolation, maceration, centrifugation, and filtration processes.

The neural crest arises from the neuro-ectoderm during embryogenesis and persists only temporarily. Early experiments already proofed pluripotent ...

Neuroscience

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 ...

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 ...

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.

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 ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

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 ...

Biology

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.
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Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

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