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 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.
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 en masse 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. 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.
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. Please click here to view a larger version of this figure.
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
2. Embryo incubation
3. Preparation of culture dishes
4. Isolation of chick embryos
5. Dissecting neural folds
6. Plating neural folds
7. Fixing and staining of cultured migratory NCCs for morphological analysis
8. Morphological assessment of cultured migratory NCCs
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 away once the yolk membranes are cut (Figure 2D,E).
Figure 2: Isolation of four to seven somite chick embryos. Fertilized eggs were incubated for 35 h. An egg was opened with blunt forceps (A), and the yolk was poured into a gloved hand (B). Excess albumin was removed (C) so that a filter paper support (D, inset) would adhere to the yolk. The embryo was cut from the yolk (D), lifted off (E), and placed into Ringer's P/S (F). Please click here to view a larger version of this figure.
After rinsing the embryo and moving to clean Ringer's P/S, neural folds were visible in the isolated embryos (Figure 3A). These folds contain premigratory cranial neural crest cells. Once excised from an embryo (Figure 3B,C) and collected (Figure 3D), isolated neural folds can be plated to create migratory NCC cultures.
Figure 3: Dissection of chick dorsal neural folds. Working in Ringer's P/S, spring scissors were used to excise neural folds. (A) Embryo dorsal view, anterior toward the top of the figure. Neural folds appear more opaque than the surrounding tissue. Midbrain neural folds lie posterior to the optic lobes (pink) and anterior to the cardiac crescent (yellow). (B) Dorsal view of the embryo after neural folds were removed, showing excision boundaries. (C) Lateral view of the embryo with neural folds removed. The dissection technique removes the dorsal neural tube, avoiding ventral and non-neural tube structures. (D) Isolated neural folds in Ringer's P/S. Scale bar = 300 μm. Please click here to view a larger version of this figure.
When neural folds were plated on FN, NCCs began to emerge from adherent neural fold explants within 3-4 h of incubation (Figure 4A), and migration was completed after approximately 20 h (Figure 4B). HNK-1 staining, which labels migratory NCCs20, was apparent in most cells in the culture (Figure 4D). A negative result occurred when neural folds failed to adhere to the coverslip, or no NCC emigrated from the explant (data not shown). NCCs were analyzed by fixing the cultures, staining for filamentous actin, and performing various measurements in ImageJ to evaluate NCC morphology and migration (Figure 5). The average area of the 69 cells analyzed was 802.11 ± 69.65 μm2, with a range from 60.27-2664.53 μm2, distributed as shown in the bar graph and violin plot (Figure 5C). Circularity (formula: circularity = 4π(area/perimeter²)), a measure which reflects the protrusiveness of a cell, ranged from 0.101-0.875, with an average of 0.38 ± 0.15. Lower values indicate an elongated shape, while a value of 1 indicates a perfect circle. As evident in the violin plot, most cells exhibit an elongated shape (Figure 5D). Another measure of cell shape is the aspect ratio (AR), which is the major axis of the cell divided by the minor axis. An AR value of 1 indicates a symmetrical shape23. Migratory NCCs in this field had an average AR of 2.13 ± 0.11, with values ranging from 1.14-5.59 (Figure 5E). Using these quantitative measures of NCC morphology, rigorous comparisons can be made between experimental conditions and different published studies.
Figure 4: Migratory NCCs emerge from cultured neural folds. Brightfield images of plated neural folds after 3 h of incubation (A) and 20 h of incubation (B). Scale bar = 200 μm. (C,D) The neural fold is largely dispersed after 20 h of incubation but residually present on the right side of these images. Scale bar = 500 μm. Migratory NCCs are visible with DAPI (C) and HNK-1 staining (D). HNK-1 immunostaining confirms that cultured cells are migratory NCCs. Please click here to view a larger version of this figure.
Figure 5: Morphological assessment of cultured migratory NCCs. (A) Phalloidin staining of filamentous actin in NCCs migrated from a neural fold after 20 h in culture. Scale bar = 50 μm. (B) Threshold image with cells appearing black and the background white. Yellow outlines surround objects counted as cells, and cells are numbered. (C–E) Graphing options to display morphological assessment data. Bar graphs and violin plots were created from measurements of the 69 cells shown in panel B to depict the area (µm2, C), circularity (D), and aspect ratio (E) of the measured cells. Bar graphs show average values with error bars representing the standard error of the mean on the left of each panel. Violin plots were created with app.rawgraphs.io. Please click here to view a larger version of this figure.
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 on high-quality eggs and reagents. In addition, due to the inherent variability of the cultures, reproducibility requires experimental repetition and quantitation.
The adhesion of neural folds to FN is essential for NCC migration in culture. Occasionally, explants will not adhere properly, and the neural fold will either float away or produce no/few migratory NCCs. This can happen when a cranial neural fold does not land cut side down; try to orient the explants in the culture vessel prior to incubation and allow them to settle for 10-15 min before moving them. However, due to fluid mixing during transfer to the incubator, some neural folds will inevitably adhere sub-optimally from time to time. When adhesion and migration are problematic for an entire experiment, this may reflect old FN (use a fresh aliquot from the -80 °C for every experiment), culture medium with expired components (store media prepared in experiment-sized aliquots at -20 °C and add fresh antibiotic at the time of use) or use of coverslips not appropriate for cell culture (for example, cells will not adhere to coverslips for histology). A contaminated incubator can also cause entire NCC culture experiments to fail.
In addition to producing dispersed NCCs in culture and allowing for analysis of migratory NCC morphology, this protocol is also a starting point for many other experiments. Explants plated on coverslips may also be processed for immunofluorescence (after section 7, step 3) to determine the subcellular localization of a protein of interest7. While analysis of morphology and migration distance are possible in fixed cultures, as described here, time-lapse imaging of NCCs during culture incubation (section 6, step 5) allows for additional data collection on directionality and persistence of migration and dynamics of cell motility (measurements of membrane protrusion, adhesion, etc.)14,24,27. Transient genetic manipulation of embryos can be achieved by electroporation18,28 of reagents (morpholinos29, DNA expression constructs, or CRISPR vectors30) at Hamburger and Hamilton stage 4+17. These electroporated embryos can then be used in the protocol to create migratory NCC cultures with loss-of-function or overexpression of genes of interest. Functional analysis can also be achieved by adding chemical inhibitors to the media of NCC cultures7,27 (section 6, step 2). Population-level profiling of NCCs through -omics level analyses requires the collection of NCCs as raw material7,11,31. While markers have been used to isolate NCCs through fluorescence-activated cell sorting31, this requires specialized flow cytometry equipment and enzymatic dissociation of cells. Following the methods described here to carefully excise dorsal neural folds (minimizing collection of the ventral neural tube and non-neural ectoderm cells) provides an inexpensive method to collect premigratory NCCs11 (section 5, step 4). Collecting NCCs after dispersal in culture (section 6, step 5) supplies a relatively pure population of naturally segregated, migratory NCCs for further analysis (HNK-1 staining, a marker of migratory NCCs, in the majority of cultured cells in Figure 4D; as in7). Finally, these variations can be used in combination. For example, immunofluorescence or time-lapse imaging can be used to evaluate the effects of gene expression manipulations or the addition of inhibitors27. Overall, this adaptable, inexpensive protocol provides the means to assess the morphology of wild-type cranial migratory NCCs in culture with multiple potential modifications to achieve various experimental goals.
The authors have nothing to disclose.
We thank Corinne A. Fairchild and Katie L. Vermillion, who participated in developing our version of the chick cranial neural fold culture protocol.
AxioObserver equipped with an LSM710 confocal scan head controlled by ZEN 3.0 SR software | Zeiss | Used alpha Plan-Apochromat 100x/1.46 Oil DIC M27 objective | |
CaCl2 | Sigma-Aldrich | C3306 | |
Chamber dishes (glass bottom, single or divided) | MatTek; Cell Vis | P35G-1.5-14-C (MatTek) X000NOJQGX (Cellvis) X000NOK1OJ (Cellvis) |
Single chamber 35 mm or 4 chamber 35 mm |
Cover glass | Carolina Biological Supply Company | 633029, 633031, 633033, 633035, 633037 | circles, 0.13–0.17 mm thickness, available in 12-25 mm diameter |
DMEM/F12 | ThermoFisher Scientific | 11320033 | Alternative for L15 media |
Egg incubator | Sportsman | 1502 | |
FBS | Life Technologies | 10437-028 | |
Fibronectin | Fisher Scientific | CB-40008A | |
Filter paper | Whatman | grade 3MM chromatography | |
Forceps (blunt) | Fisher Scientific; Thomas Scientific | 08-890 (Fisher);1141W97 (Thomas) | |
Forceps (fine) | Fine Science Tools | 11252-20 | Dumont #5 |
Image J | https://fiji.sc/ | Free image analysis software | |
KCl | Sigma-Aldrich | P3911 | |
KH2PO4 | Sigma-Aldrich | P0662 | |
L15 media | Invitrogen | 11415064 | |
L-glutamine | Invitrogen | 25030 | |
Mounting Media (Vectashield or ProLong Gold) | Vector Laboratories; Thermofisher Scientific | H-1700 (Vectashield); P36930 (ProLong Gold) | |
Na2HPO4 | Sigma-Aldrich | S9638 | |
NaCl | Sigma-Aldrich | S9888 | |
Paraformaldehyde | Sigma-Aldrich | P6148 | |
Penicillin/streptomycin | Life Technologies | 15140-148 | 10,000 Units/mL Penicillin; 10,000 mg/mL Streptomycin |
Petri Dishes | VWR (or similar) | 60 mm, 100 mm | |
Phalloidin | Sigma-Aldrich | P1951 | multiple flurophores available |
Pin holder | Fine Science Tools | 26016-12 | For tungsten needle (alternative for spring scissors) |
Scissors (dissection) | Fine Science Tools | 14061-10 | |
Spring Scissors | Fine Science Tools | 15000-08 | 2.5 mm cutting edge (alternative for tungsten needle) |
Sylgard | Krayden | Sylgard 184 | |
Syringe Filters | Sigma-Aldrich | SLGVM33RS | Millex-GV Syringe Filter Unit, 0.22 µm, PVDF, 33 mm, gamma sterilized |
Tissue culture dishes | Sarstedt | 83-3900 | 35 mm culture dishes for bulk neural fold cultures |
Triton X-100 | Sigma-Aldrich | X100 | |
Tungsten wire | Variety of sources | 0.01" diameter for tungsten needle (alternative for spring scissors) |