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

Summary

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.

Abstract

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 cartilage^5-13, melanocytes^11,14-20, neurons and glia^21-32. 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 embryo^11,33-37, 2) signals from neighboring cells as they migrate^38-44, and 3) the microenvironment of their ultimate destination within the embryo^45,46. 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 axis^2,47. 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 sample^2,47. 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 antibody^48-56 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 molecules^57,58, or genetic manipulation via electroporation of expression plasmids^59-61, 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

Protocol

1. Incubate chick and quail eggs to the desired stage

For HH9 embryos, typical incubation times range from 29-33 hours at 38 °C.⁶³

1. Wash any debris from the eggs with tepid water.
2. 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.
3. Place in 38 °C humidified incubator. Turn on rocking function.

2. Prepare eggs for windowing and dissection

1. Remove eggs from incubator, and sterilize the tops with 70% ethanol. It is best to spr....

Discussion

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 axis^21,67-69. 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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chick eggs	Various
Quail eggs	Various
Egg incubator (Digital Readout 1502 Sportsman Incubator w/Humidity 110-120 Volt AC)	Poultry Supply	1502
Dumont AA forceps, Inox Epoxy-coated	Fine Science Tools	11210-10
Scotch tape	Any Supplier
Curved Iris forceps	Fine Science Tools	11065-07
India ink	Any Supplier
Pen/Strep (Penicillin, Streptomycin) Solution	VWR international	101447-068
Clear Packing tape	Any Supplier
Needle pulling apparatus	Narishige International	PE-21
Pulled glass needle, made from 1.5-1.8 x 100mm borosilicate glass capillary tube	Kimble Chase	34500 99
Pulled glass pipette, made from 5¾" Pasteur pipette	Fisher Scientific	13-678-6A
Mouth pipette apparatus (aspirator tube assembly for calibrated microcapillary pipette)	Sigma-Aldrich	A5177-52A
Dumont #5 forceps	Fine Science Tools	11251-30
Tungsten wire, 0.1mm diameter	VWR international	AA10404-H2
Needle holders (Nickel-plated pin holder)	Fine Science Tools	26018-17
QCPN antiserum	Developmental Studies Hybridoma Bank	QCPN
Alexa Fluor secondary antibody (e.g., Alexa Fluor 594 goat anti-mouse IgG1)	Invitrogen	A21125
Ringer’s Solution (2L): 14.4g NaCl 0.34g CaCl2 0.74g KCl 0.230g Na2HPO4 0.04g KH2PO4 ddH2O to 2L Filter and autoclave	Fisher Scientific	7647-14-5 10043-52-4 7447-40-7 7558-79-4 7778-77-0