The authors thank members of the Lwigale laboratory for critique of the manuscript. SLG is supported by a Ruth L. Kirschstein NRSA Fellowship from the National Eye Institute (F32 EY02167301). PYL is supported by the National Eye Institute (EY018050).

1. Incubate chick and quail eggs to the desired stage
For HH9 embryos, typical incubation times range from 29-33 hours at 38 &deg;C.63
<ol>
	<li>Wash any debris from the eggs with tepid water.</li>
	<li>Arrange chicken eggs on tray horizontally. Mark the side facing up with pencil; this will correspond to the region where the embryo will be localized. Incubate quail eggs blunt end up.</li>
	<li>Place in 38 &deg;C humidified incubator. Turn on rocking function.</li>
</ol>
2. Prepare eggs for windowing and dissection
<ol>
	<li>Remove eggs from incubator, and sterilize the tops with 70% ethanol. It is best to spray on the ethanol and wipe it off quickly with a paper towel or Kimwipe to avoid any absorption of ethanol through the eggshell.</li>
	<li>Place chicken egg onto an individual egg holder (we use a Petri dish lined with folded paper towels, e.g., "Kimwipes"). Using AA forceps, tap a small hole into the upper surface of the eggshell at the pointed end of the egg.</li>
	<li>Remove 1.5-3mL of light albumin from the chicken egg with an 18&frac12; G hypodermic needle and 5 mL syringe. It is best to insert the needle vertically into the hole, with the bevel facing the pointy end of the egg (Figure 2A). This way, when albumin is withdrawn, there is little risk of accidentally damaging the yolk via suction. Discard the albumin. This step will lower the yolk and embryo within the egg to allow for a window to be opened in the eggshell.</li>
	<li>Wipe the hole with 70% ethanol as described above and seal it with Scotch tape. It is important that the eggshell surrounding the hole is completely devoid of albumin and dry or the adhesive will not seal.</li>
	<li>With the AA forceps, tap another hole into the marked upper surface of the horizontal egg, not quite at the apex. Take care not to let the forceps go too far through the eggshell to avoid damaging the yolk or embryo.</li>
	<li>Insert one side of the curved iris forceps into the hole parallel to the eggs shell. Pinching down on the shell, working in a circular motion to break a ~2 cm in diameter window in the chicken egg shell. Discard the removed eggshell. Alternatively, cover the upper surface of the egg with packing tape and use a pair of scissors to cut out the window. This minimizes the chance of eggshell debris falling into the egg. The embryo should appear as an opaque disc on top of the yolk. Discard any unfertilized eggs (identified by small white spot on surface of yolk, or absence of blastoderm).</li>
	<li>Inject a small amount of India ink (diluted 1:10 in sterile Ringer's solution containing "Pen/Strep" antibiotic: final concentration 100 &mu;g/mL penicillin and 100 &mu;g/mL streptomycin) beneath the blastoderm to assist with staging the embryo. Use a 1 mL syringe and 26&frac12; G hypodermic needle, bent into a 45&deg; angle at the base of the needle, with the bevel facing up; alternatively, one may use a pulled glass Pasteur pipet and mouth pipetting apparatus (wear eye protection when bending hypodermic needle or breaking glass needle). Puncture the yolk membrane outside the perimeter of the blastoderm and slide the tip of the needle beneath the embryo, close to the surface of the yolk but not in the embryonic layers (Figure 2B). Inject just enough ink to outline the embryo, then carefully withdraw the needle (Figure 2C). Injecting too much ink can lead to embryo death. It is important not to inject any air bubbles beneath the embryo, as this can be a source for contamination. NB: All Ringer's solution used in this procedure is sterilized and contains Pen/Strep as defined above. Sterile phosphate buffered saline (PBS) is also an acceptable alternative to Ringer's solution in all steps of this protocol.</li>
	<li>Stage the embryo according to Hamburger and Hamilton63 and record the stage on the eggshell in permanent ink or pencil. Stages are best assessed under stereomicroscopy, with fiber optic "gooseneck" light sources which have a limited heat load.</li>
	<li>Apply 2-3 drops warm sterile Ringer's solution to the surface of the embryo to prevent dehydration and contamination. Temporarily seal the window with parafilm stretched over the surface of the egg.</li>
	<li>Repeat steps 2.2-2.9 with all chick eggs before beginning the transplant experiments.</li>
	<li>Since quail embryos are only used for donor tissues, in ovo steps 2.2-2.9 may be omitted. For ex ovo preparation of donor embryos, quail eggs are incubated blunt side up and opened in this region with curved forceps to reveal the embryo. Using dissecting scissors, make four cuts in the shape of a square through the vitelline membrane and blastoderm outside the embryo (make sure that all cuts meet). Using a curved iris forceps, gently grasp one cut edge and lift the embryo from the egg and transfer it to Ringers solution in a Petri dish. Alternatively, one may use the curved iris forceps to lift the excised embryo from beneath, or an embryo spoon or plastic transfer pipette cut through the wider part of the cylinder, may be used to transfer the embryo out of the egg. Gently remove the vitelline membrane with #5 forceps. Collect the remaining quail embryos as above, and stage them under a dissecting microscope.</li>
</ol>
3. Prepare the host embryo to receive the graft
<ol>
	<li>Remove parafilm from the host (chick) embryo and using a sharpened tungsten needle or #5 forceps, tear a small hole in the vitelline membrane at the desired region of the neural tube (Figure 3A).</li>
	<li>Add a drop of Ringer's solution to the embryo. Take care to keep the embryo completely submerged in Ringer's solution during the entire procedure to prevent dehydration of the tissue. Keep adding drops of Ringer's solution to the surface of the embryo via transfer pipette if necessary.</li>
	<li>Using a pulled glass needle, carefully make rostral and caudal transverse incisions corresponding to the length and region of interest in the dorsal neural tube. (Pulled glass needles are generated from silicon glass capillary tubes which have been pulled under heat with a needle pulling apparatus.) For unilateral grafts the incisions should only extend to the lumen of the dorsal neural tube, and for bilateral grafts, make the incisions across the entire dorsal neural tube. Then cut bilaterally between the dorsal neural tube and the paraxial mesoderm.</li>
	<li>Carefully separate the excised explant from the neural tube then remove it from the embryo by aspirating into a glass micropipette (Figure 3B).</li>
</ol>
4. Prepare the donor graft tissue
<ol>
	<li>Choose a stage-matched quail embryo. Hold the embryo down with AA forceps, remove the vitelline membrane, and excise a similar-sized region of neural tube as described above (3.3-3.4) (Figure 3A'-B'). Depending on the needs of your experiment, this region may be a complementary region of the host (chick) neural tube or from a different region along the rostro-caudal axis. (NB: for larger regional grafts, including complete neural tube grafts, researchers may wish to add a protease digestion at this step to remove any adherent mesenchymal tissue from the donor tissue prior to transplantation. However, for small dorsal neural tube grafts at early stages, as is detailed here, protease digestion is not necessary as the neural tube is easily separated from adjacent mesenchyme by strictly surgical techniques.)</li>
</ol>
5. Graft the tissue
<ol>
	<li>Aspirate the donor explant into a glass micropipette containing Ringer's solution. Be careful not to introduce any bubbles into the pipette.</li>
	<li>Transfer the explant adjacent to the excised region of the chick host. Using pulled glass needle, orient the explant and gently guide it into the ablated region (Figure 3C).</li>
	<li>Carefully add a few drops of Ringer's solution to the embryo to prevent dehydration. Take care to avoid dropping liquid directly on the graft site as this may dislodge the graft.</li>
	<li>Seal the window with packing tape. Make sure that the tape seals the entire region of the windowed egg. This prevents dehydration and contamination of the embryo during subsequent incubation.</li>
	<li>Return the egg to the incubator until the desired stage. Make sure that the "rocking" function is turned off while incubating the chimeras. Eggs may be gently turned by hand two times a day as this may increase their viability if later stages of development are targeted.</li>
</ol>
6. Prepare chimeric embryos for sectioning
<ol>
	<li>At the experimental endpoint, cut away the tape covering the window using fine scissors.</li>
	<li>Grasp the edge of the blastoderm with curved iris forceps (easiest to do with serrated tips), then cut the embryo away from the yolk with 4 large cuts outside the boundaries of the blastoderm.</li>
	<li>Transfer the embryo to a Petri dish containing Ringer's solution. Take care to minimize exposure of the embryo to air in order to avoid dehydration of the tissue.</li>
	<li>Under stereomicroscopy, gently separate the vitelline membrane from the blastoderm using #5 forceps.</li>
	<li>Carefully arrange the embryo in the dish so that the embryo is in the same orientation as it was in the egg, with surrounding membranes laid out flat.</li>
	<li>Use dissecting needles made from tungsten wires64 to cleanly remove the surrounding blastoderm from the embryo by arranging the needle at the boundary of the embryo and extraembryonic tissues, with the length of the tungsten wire parallel to the bottom of the dish. Use a gentle sawing motion to cut through the tissues.</li>
	<li>Carefully remove the amnion with #5 forceps. At this point you may, wish to either further dissect the region of your interest, or leave the embryo whole.</li>
	<li>Transfer the embryo to ice cold 4% paraformaldehyde and rock overnight at 4 &deg;C.</li>
	<li>Prepare samples and embed for cryo- or paraffin sectioning.</li>
</ol>
Trace the grafted tissue within the host embryo. There are several techniques for identifying quail tissue within a chick embryo, including detection of quail nucleoli by hematoxylin staining (quail nuclei have very large, darker staining inclusions than chick nuclei), the Feulgen-Rossenbeck reaction, acridine-orange or biz-benzamide stain combined with electron microscopy, or immunolabeling for quail-specific antigens3,47,65,66. Here we use QCPN antigen and standard whole-mount or section immunofluorescence techniques to identify quail neural crest cells in quail-chick chimeras (Figure 4). This technique provides the most flexibility in experimental design, as the QCPN immunofluorescence may also be combined with other antibodies to identify differentiated cells derived from the donor (quail) tissue. Use standard whole-mount or section immunofluorescence techniques to label quail-derived cells in chimeras.
7. Representative Results
A representative image of the grafted region of the neural tube after 6h of re-incubation (to HH11) shows expected incorporation of the grafted donor (quail) tissue into the host (chick) neural tube (Figure 4A). Embryos showing incomplete integration of the graft, or asymmetric development of the cranial region or somites after reincubation should be discarded.
Cross section through the grafted region at HH11 show NCCs labeled with HNK-1 migrating laterally away from the neural tube. Quail cells contributing to the NCC migratory stream, and to the neural tube, are clearly labeled with QCPN (Figure 4B).
At later stages, quail NCC-derived cells can be traced to their final target tissue. QCPN labeled cells are interspersed within the chick embryo mesenchyme of the maxillary process at E5 (Figure 4C).
The QCPN antibody can be easily combined with other antibodies to examine the differentiation of quail-derived NCCs in the host environment. Quail NCC-derived trigeminal sensory neurons are labeled by QCPN and Tuj1 antibodies (Figure 4D).
<img alt="Figure 1" src="/files/ftp_upload/3622/3622fig1.jpg" /> 
Figure 1. Overview of experimental procedure and necessary instruments. A) Host (chick) and donor (quail) embryos should be stage matched. To label the NCC stream that contributes to the trigeminal ganglion and maxillo-mandibular region, HH9 is ideal. At HH9, the neural tube is beginning to close in the rostral region, but is not yet completely sealed. The gray line in the diagrams represents the midline of the closing neural tube. The dotted white lines indicate the cut lines for excising a midbrain region of the right side of the neural tube from host and donor embryos. The excised region of the host embryo is discarded and the donor tissue transplanted in to generate the chimeric embryo. B) Necessary instruments include: a) 5mL syringe with 18&frac12; G hypodermic needle, b) 1 mL syringe with 26&frac12; G hypodermic needle bent at 45&deg; angle, c) India ink, d) clear packing tape, e) glass pipette with mouth pipetting apparatus, f) 60 mm-Petri dish, g) AA forceps, h) curved iris forceps with serrated tips, i) #5 forceps, j) fine scissors, k) sharpened tungsten wire, l) pulled glass needle, m) parafilm squares.
<img alt="Figure 2" src="/files/ftp_upload/3622/3622fig2.jpg" /> 
Figure 2. Preparation of eggs and embryos. A) Cross-sectional diagram of the egg, including ideal insertion point of 18&frac12; G hypodermic needle for withdrawal of light albumin. B) After withdrawing ~3mL of light albumin, the yolk and embryo lowers within the egg, allowing the researchers to cut a "window" (arrows) in the eggshell to access the embryo. India ink, diluted 1:10 in sterile Ringer's solution, can then be injected beneath the blastoderm to provide contrast for easy staging of the embryos. C) Chick embryo after inking.
<img alt="Figure 3" src="/files/ftp_upload/3622/3622fig3.jpg" /> 
Figure 3. Schematic and examples of HH stage 9 donor and host embryos at each step of the grafting procedure. A) Chick host embryo. Dashed lines in diagram indicate where the vitelline membrane should be torn to access the cranial region for grafting. Once torn the triangular flap of vitelline membrane should be peeled caudally. Box in image indicates inset used in (B-C). A') Quail donor embryo. Image is of donor embryo excised from egg and placed into Petri dish for dissection of the graft tissue. Box in image indicates inset used in (B'). Dashed lines in schematic diagram (A') indicate where the vitelline and yolk membranes should be cut in order to remove the embryo from the egg. B) Host embryo with unilateral midbrain region of neural tube excised (white arrows) awaiting graft. Dotted line in schematic diagram indicates where cuts should be made to excise the neural tube in the midbrain region. B') Donor embryo with dorsal neural tube graft tissue excised (graft tissue indicated by black arrowheads). Dotted line in diagram indicates where cuts should be made in the quail embryo to excise the donor tissue for unilateral grafting of the midbrain region of the neural tube. C) Chimeric embryo after grafting of quail dorsal neural tube explant into the chick midbrain region.
<img alt="Figure 4" src="/files/ftp_upload/3622/3622fig4.jpg" /> 
Figure 4. Immunofluorescence detecting quail-specific nuclear antigen QCPN in donor-derived cells in the chimeric embryo. A) Whole-mount image of HH11 chimera (grafted at HH9) showing QCPN staining in red, and HNK-1 staining (a marker of migrating NCCs) in green. 10X magnification, dorsal view. B) Cross section through embryo in (A), showing QCPN-positive quail derived cells in the neural tube and migratory NCC co-stained with HNK-1 (green). 40X magnification. C) Cross-section through maxillary process of E5 chimera (incubated for 3 days post graft), showing QCPN-positive NCC-derived cells (red) which have migrated from the grafted region of the neural tube to their final location in the embryo. 10X magnification. D) Section through E5 chimera showing contribution of quail derived NCC to the trigeminal ganglion (red) and differentiation into TuJ1-positive neurons (green). 40X magnification. *, NCCs; MP, maxillary process; NT, neural tube; TG, trigeminal ganglion.

The authors have nothing to disclose.

The grafting of quail neural tube into host chick embryos described here is a straightforward and inexpensive technique for tracing specific subpopulations of migrating NCCs emanating from different regions along the rostro-caudal axis21,67-69. This technique takes advantage of the ease of access to avian embryos (as compared to mammalian embryos) and may be combined with other techniques, such as tissue ablation, injection of inhibitory molecules, or genetic manipulation via electroporation of expression plas...

<table><tbody><tr><td>Chick eggs</td><td>Various</td><td></td><td></td></tr><tr><td>Quail eggs</td><td>Various</td><td></td><td></td></tr><tr><td>Egg incubator (Digital Readout 1502 Sportsman Incubator w/Humidity 110-120 Volt AC)</td><td><a href="http://www.poultrysupply.com" target="_blank"><img src="/img/new_window.png" /> Poultry Supply</a></td><td>1502</td><td></td></tr><tr><td>Dumont AA forceps, Inox Epoxy-coated</td><td>Fine Science Tools</td><td>11210-10</td><td></td></tr><tr><td>Scotch tape</td><td>Any Supplier</td><td></td><td></td></tr><tr><td>Curved Iris forceps</td><td>Fine Science Tools</td><td>11065-07</td><td></td></tr><tr><td>India ink</td><td>Any Supplier</td><td></td><td></td></tr><tr><td>Pen/Strep (Penicillin, Streptomycin) Solution </td><td>VWR international</td><td>101447-068</td><td></td></tr><tr><td>Clear Packing tape</td><td>Any Supplier</td><td></td><td></td></tr><tr><td>Needle pulling apparatus</td><td>Narishige International</td><td>PE-21</td><td></td></tr><tr><td>Pulled glass needle, made from 1.5-1.8 x 100mm borosilicate glass capillary tube</td><td>Kimble Chase</td><td>34500 99</td><td></td></tr><tr><td>Pulled glass pipette, made from 5&amp;#190;&quot; Pasteur pipette</td><td>Fisher Scientific</td><td>13-678-6A</td><td></td></tr><tr><td>Mouth pipette apparatus (aspirator tube assembly for calibrated microcapillary pipette)</td><td>Sigma-Aldrich</td><td>A5177-52A</td><td></td></tr><tr><td>Dumont #5 forceps</td><td>Fine Science Tools</td><td>11251-30</td><td></td></tr><tr><td>Tungsten wire, 0.1mm diameter</td><td>VWR international</td><td>AA10404-H2</td><td></td></tr><tr><td>Needle holders (Nickel-plated pin holder)</td><td>Fine Science Tools</td><td>26018-17</td><td></td></tr><tr><td>QCPN antiserum</td><td>Developmental Studies Hybridoma Bank</td><td>QCPN</td><td></td></tr><tr><td>Alexa Fluor secondary antibody (e.g., Alexa Fluor 594 goat anti-mouse IgG1)</td><td>Invitrogen</td><td>A21125</td><td></td></tr><tr><td>Ringer&amp;rsquo;s Solution (2L): &lt;ul&gt; &lt;li&gt;14.4g NaCl&lt;/li&gt; &lt;li&gt;0.34g CaCl&lt;sub&gt;2&lt;/sub&gt;&lt;/li&gt; &lt;li&gt;0.74g KCl&lt;/li&gt; &lt;li&gt;0.230g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;&lt;/li&gt; &lt;li&gt;0.04g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;&lt;/li&gt; &lt;li&gt;ddH&lt;sub&gt;2&lt;/sub&gt;O to 2L&lt;/li&gt; &lt;li&gt;Filter and autoclave&lt;/li&gt; &lt;/ul&gt;</td><td>Fisher Scientific</td><td>&lt;ul&gt; &lt;li&gt;7647-14-5&lt;/li&gt; &lt;li&gt;10043-52-4&lt;/li&gt; &lt;li&gt;7447-40-7&lt;/li&gt; &lt;li&gt;7558-79-4&lt;/li&gt; &lt;li&gt;7778-77-0&lt;/li&gt; &lt;/ul&gt;</td><td></td></tr></tbody></table>

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

Watch this Scientific Journal Video about Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation at JoVE.com

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

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. ...

analysis-neural-crest-migration-differentiation-cross-species

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22.7K Views.  Rice University. 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.

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

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 ...

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 ...

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 ...

Developmental Biology

Creating Avian Forebrain Chimeras to Assess Facial Development

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the strengths of this model system is that the effect of, and interaction among, tissues can be directly assessed in chimeric embryos. We have previously shown that signals from the forebrain contribute to facial morphogenesis by regulating the shape of the expression domain of Sonic hedgehog (SHH) in the Frontonasal Ectodermal Zone (FEZ). In this article, the method of generating the forebrain chimeras and provide illustrations of the outcomes of these experiments is described.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of basal forebrain&#160;during craniofacial development.

The avian embryo has been used as a model system for more than a century and has led to fundamental understanding of vertebrate development. One of the ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Biology

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

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

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

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

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 ...

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.

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

Summary

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Chapters in this video

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

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

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

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

Creating Avian Forebrain Chimeras to Assess Facial Development

Xenotransplantation of Human Stem Cells into the Chicken Embryo

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

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

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