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1Department of Biochemistry and Cell Biology, Rice University
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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.
Griswold, S. L., Lwigale, P. Y. Analysis of Neural Crest Migration and Differentiation by Cross-species Transplantation. J. Vis. Exp. (60), e3622, doi:10.3791/3622 (2012).
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
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
2. Prepare eggs for windowing and dissection
3. Prepare the host embryo to receive the graft
4. Prepare the donor graft tissue
5. Graft the tissue
6. Prepare chimeric embryos for sectioning
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).
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 18½ G hypodermic needle, b) 1 mL syringe with 26½ G hypodermic needle bent at 45° 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.
Figure 2. Preparation of eggs and embryos. A) Cross-sectional diagram of the egg, including ideal insertion point of 18½ 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.
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.
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 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 plasmids, to experimentally examine the response of specific migratory NCC populations to different developmental cues within the embryos47,69.
Quail-chick chimeras are a powerful tool for examining NCC migration and differentiation, and this technique may be adapted to include transplantation of other tissues besides the neural tube. Grafting of quail tissue into the chick embryo is a well-established technique13,21,70-76, but is best learned by visual demonstration, as the microdissection of small regions of neural tubes require very fine motor skills.
Because quail-derived cells can be easily distinguished from host chick cells via immunostaining with the quail-specific antibody QCPN, the developmental potential of NCCs arising from specific regions of the neural tube can be inferred from differentiation of donor (quail) cells at the experimental end point; for instance, the presence of QCPN positive cells in the periocular region and cornea, after grafting quail neural tube into the midbrain region of chick embryos at HH9 indicates that migratory neural crest from this region give rise to corneal endothelium and stroma77. Neural tube grafts at HH9-10 reveal NCC contribution to the trigeminal ganglion and branchial arches. Also, quail-derived sensory neurons can be tracked using another quail-neuron specific (QN) antibody78,79.
The authors have nothing to disclose.
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).
|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):
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