Summary

تحليل للهجرة كريست العصبية والتمايز بواسطة الصليب الأنواع زرع

Published: February 07, 2012
doi:

Summary

يوصف هذا النهج لتحليل الهجرة والمصير النهائي من الطيور الخلايا العصبية في قمة السمان، فرخ أجنة خيالية. هذا الأسلوب هو أسلوب بسيط ومباشر لتعقب الخلايا العصبية خلال قمة للهجرة والتمايز التي يصعب على خلاف ذلك للتمييز في جنين الفرخ unmanipulated.

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 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

Protocol

1. احتضان الفرخ والبيض السمان إلى المرحلة المطلوبة لHH9 الأجنة، مرات حضانة نموذجية تتراوح بين 29-33 ساعة عند 38 درجة مئوية 63 غسل أي حطام من البيض مع الماء الفاتر. <li style=";text-align:rig…

Discussion

ووصف عمليات ترقيع من الأنبوب العصبي السمان في افراخ الدجاج المضيف هنا هي تقنية بسيطة وغير مكلفة لتعقب مجموعات سكانية فرعية محددة من الهجرة البلدان المساهمة الصافية النابعة من مناطق مختلفة على طول المحور rostro-الذيلية 21،67-69. هذه التقنية تستفيد من سهولة الحصول عل?…

Disclosures

The authors have nothing to disclose.

Acknowledgements

الكتاب أشكر أعضاء مختبر Lwigale للنقد من المخطوطة. ويدعم SLG من قبل L. روث زمالة NRSA كيرشتاين من المعهد الوطني للعيون (F32 EY02167301). ويدعم PYL من قبل المعهد الوطني للعيون (EY018050).

Materials

Reagent Company Catalog number
Chick eggs Various – we use Texas A&M University’s Poultry Science Department, TX.  
Quail eggs Various – we use Ozarks Egg Company, MO.  
Egg incubator (Digital Readout 1502 Sportsman Incubator w/Humidity 110-120 Volt AC) www.poultrysupply.com 1502
Dumont AA forceps, Inox Epoxy-coated Fine Science Tools 11210-10
Scotch tape Any office supply store  
Curved Iris forceps Fine Science Tools 11065-07
India ink Any art supply store  
Pen/Strep (Penicillin, Streptomycin) Solution VWR International 101447-068
Clear Packing tape Any office supply store  
Needle pulling apparatus Narashige, Japan 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, University of Iowa 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
All reagents from Fisher Scientific
  • 7647-14-5
  • 10043-52-4
  • 7447-40-7
  • 7558-79-4
  • 7778-77-0

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Cite This Article
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).

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