Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

Summary

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.

Abstract

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.

Introduction

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 population

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

1. Prepare Ringer'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....

Results

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.......

Discussion

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 methods^24,25,26. While this in vitro method is relatively simple, consistent results depend o.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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)