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Neuroscience

Анализ нейронной миграции Крест и дифференциация по межвидовой трансплантации

Published: February 07, 2012
doi:
10.3791/3622
,
1Department of Biochemistry and Cell Biology,Rice University

Summary

Подход к анализу миграции и в конечном итоге судьба птичьего клеток нервного гребня в перепелиных-химерных эмбрионов куриных описано. Этот метод является простым и техника для отслеживания клеток нервного гребня во время миграции и дифференциации, которые иначе трудно различить в unmanipulated куриного эмбриона.

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. Инкубируйте куриных и перепелиных яиц желаемый этап Для HH9 эмбрионов, типичное время инкубации составляет от 29-33 часов при 38 ° C. 63 Смойте остатки из яиц с теплой водой. Упорядочить куриные яйца на подносе по горизонтали. Отметить вверх с карандашом, это …

Discussion

Прививки перепела нервной трубки эмбриона в принимающей куриных описанный здесь простой и недорогой способ для отслеживания конкретных субпопуляций мигрирующих НКЦ, исходящие из разных регионов по-ростро хвостовой оси 21,67-69. Эта техника использует легкость доступа к птичьему э…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Авторы выражают благодарность членам Lwigale лаборатории критику рукописи. SLG поддерживается Рут Л. Kirschstein НРСА стипендии от Национального института глаза (F32 EY02167301). Пыл при поддержке Национального института глаза (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
DOWNLOAD MATERIALS LIST

References

  1. Le Douarin, G., Renaud, D. Morphologic and physiologic study of the differentiation in vitro of quail embryo precardial mesoderm. Bull. Biol. Fr. Belg. 103 (3), 453-468 (1969).
  2. Teillet, M. A., Ziller, C., Le Douarin, N. M. Quail-chick chimeras. Methods. Mol. Biol. 461, 337-350 (2008).
  3. Le Douarin, N. A biological cell labeling technique and its use in expermental embryology. Dev. Biol. 30 (1), 217-222 (1973).
  4. Noden, D. M. An analysis of migratory behavior of avian cephalic neural crest cells. Dev. Biol. 42 (1), 106-130 (1975).
  5. Johnston, M. C. A radioautographic study of the migration and fate of cranial neural crest cells in the chick embryo. Anat. Rec. 156 (2), 143-155 (1966).
  6. Noden, D. M. The control of avian cephalic neural crest cytodifferentiation. I. Skeletal and connective tissues. Dev. Biol. 67 (2), 296-312 (1978).
  7. Oka, K. The role of TGF-beta signaling in regulating chondrogenesis and osteogenesis during mandibular development. Dev. Biol. 303 (1), 391-404 (2007).
  8. Chai, Y. Fate of the mammalian cranial neural crest during tooth and mandibular morphogenesis. Development. 127 (8), 1671-1679 (2000).
  9. Lengele, B., Schowing, J., Dhem, A. Embryonic origin and fate of chondroid tissue and secondary cartilages in the avian skull. Anat. Rec. 246 (3), 377-393 (1996).
  10. Le Douarin, N. M., Ziller, C., Couly, G. F. Patterning of neural crest derivatives in the avian embryo: in vivo and in vitro studies. Dev. Biol. 159 (1), 24-49 (1993).
  11. Lallier, T. E. Cell lineage and cell migration in the neural crest. Ann. N.Y. Acad. Sci. 615, 158-171 (1991).
  12. Nakamura, H. Mesenchymal derivatives from the neural crest. Arch. Histol. Jpn. 45 (2), 127-138 (1982).
  13. Le Lievre, C. S., Le Douarin, N. M. Mesenchymal derivatives of the neural crest: analysis of chimaeric quail and chick embryos. J. Embryol. Exp. Morphol. 34 (1), 125-154 (1975).
  14. Rawles, M. E. The Development of Melanophores from Embryonic Mouse Tissues Grown in the Coelom of Chick Embryos. Proc. Natl. Acad. Sci. U.S.A. 26 (12), 673-680 (1940).
  15. Rawles, M. E. The Pigment-Forming Potency of Early Chick Blastoderms. Proc. Natl. Acad. Sci. U.S.A. 26 (1), 86-94 (1940).
  16. Mosher, J. T. Intrinsic differences among spatially distinct neural crest stem cells in terms of migratory properties, fate determination, and ability to colonize the enteric nervous system. Dev. Biol. 303 (1), 1-15 (2007).
  17. Dupin, E., Le Douarin, N. M. Development of melanocyte precursors from the vertebrate neural crest. Oncogene. 22 (20), 3016-3023 (2003).
  18. Faraco, C. D., Vaz, S. A., Pastor, M. V., Erickson, C. A. Hyperpigmentation in the Silkie fowl correlates with abnormal migration of fate-restricted melanoblasts and loss of environmental barrier molecules. Dev. Dyn. 220 (3), 212-225 (2001).
  19. Selleck, M. A., Bronner-Fraser, M. Avian neural crest cell fate decisions: a diffusible signal mediates induction of neural crest by the ectoderm. Int. J. Dev. Neurosci. 18 (7), 621-627 (2000).
  20. Stocker, K. M., Sherman, L., Rees, S., Ciment, G. Basic FGF and TGF-beta 1 influence commitment to melanogenesis in neural crest-derived cells of avian embryos. Development. 111 (2), 635-645 (1991).
  21. Le Douarin, N. M., Teillet, M. A. Experimental analysis of the migration and differentiation of neuroblasts of the autonomic nervous system and of neurectodermal mesenchymal derivatives, using a biological cell marking technique. Dev. Biol. 41 (1), 162-184 (1974).
  22. Noden, D. M. The control of avian cephalic neural crest cytodifferentiation. II. Neural tissues. Dev. Biol. 67 (2), 313-329 (1978).
  23. Barraud, P. Neural crest origin of olfactory ensheathing glia. Proc. Natl. Acad. Sci. U.S.A. 107 (49), 21040-21045 (2010).
  24. Li, H. Y., Say, E. H., Zhou, X. F. Isolation and characterization of neural crest progenitors from adult dorsal root ganglia. Stem Cells. 25 (8), 2053-2065 (2007).
  25. Carney, T. J. A direct role for Sox10 in specification of neural crest-derived sensory neurons. Development. 133 (23), 4619-4630 (2006).
  26. Maro, G. S. Neural crest boundary cap cells constitute a source of neuronal and glial cells of the PNS. Nat. Neurosci. 7 (9), 930-938 (2004).
  27. Bronner-Fraser, M. Molecular analysis of neural crest formation. J. Physiol. Paris. 96 (1-2), 3-8 (2002).
  28. Paratore, C., Goerich, D. E., Suter, U., Wegner, M., Sommer, L. Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling. Development. 128 (20), 3949-3961 (2001).
  29. Britsch, S. The transcription factor Sox10 is a key regulator of peripheral glial development. Genes Dev. 15 (1), 66-78 (2001).
  30. Bronner-Fraser, M. Origin of the avian neural crest. Stem Cells. 13 (6), 640-646 (1995).
  31. Jessen, K. R., Mirsky, R. Neural development. Fate diverted. Curr. Biol. 4 (9), 824-827 (1994).
  32. Le Douarin, N., Dulac, C., Dupin, E., Cameron-Curry, P. Glial cell lineages in the neural crest. Glia. 4 (2), 175-184 (1991).
  33. Chan, W. Y., Cheung, C. S., Yung, K. M., Copp, A. J. Cardiac neural crest of the mouse embryo: axial level of origin, migratory pathway and cell autonomy of the splotch (Sp2H) mutant effect. Development. 131 (14), 3367-3379 (2004).
  34. Bronner-Fraser, M. Segregation of cell lineage in the neural crest. Curr. Opin. Genet. Dev. 3 (4), 641-647 (1993).
  35. Peters-vander Sanden, M. J., Luider, T. M., vander Kamp, A. W., Tibboel, D., Meijers, C. Regional differences between various axial segments of the avian neural crest regarding the formation of enteric ganglia. Differentiation. 53 (1), 17-24 (1993).
  36. Kuratani, S., Bockman, D. E. Capacity of neural crest cells from various axial levels to participate in thymic development. Cell Tissue Res. 263 (1), 99-105 (1991).
  37. Leblanc, G. G., Epstein, M. L., Bronner-Fraser, M. E. Differential development of cholinergic neurons from cranial and trunk neural crest cells in vitro. 137 (2), 318-330 (1990).
  38. Golding, J. P., Trainor, P., Krumlauf, R., Gassmann, M. Defects in pathfinding by cranial neural crest cells in mice lacking the neuregulin receptor ErbB4. Nat. Cell. Biol. 2 (2), 103-109 (2000).
  39. Kulesa, P. M., Bailey, C. M., Kasemeier-Kulesa, J. C., McLennan, R. Cranial neural crest migration: new rules for an old road. Dev. Biol. 344 (2), 543-554 (2009).
  40. Lwigale, P. Y., Bronner-Fraser, M. Semaphorin3A/neuropilin-1 signaling acts as a molecular switch regulating neural crest migration during cornea development. Dev. Biol. 336 (2), 257-265 (2009).
  41. Killian, O. l. e. s. n. i. c. k. y., Birkholz, E. C., A, D., Artinger, K. B. A role for chemokine signaling in neural crest cell migration and craniofacial. Dev. Biol. 333 (1), 161-172 (2009).
  42. Gammill, L. S., Gonzalez, C., Bronner-Fraser, M. Neuropilin 2/semaphorin 3F signaling is essential for cranial neural crest migration and trigeminal ganglion condensation. Dev. Neurobiol. 67 (1), 47-56 (2007).
  43. Osborne, N. J., Begbie, J., Chilton, J. K., Schmidt, H., Eickholt, B. J. Semaphorin/neuropilin signaling influences the positioning of migratory neural crest cells within the hindbrain region of the chick. Dev. Dyn. 232 (4), 939-949 (2005).
  44. Kanzler, B., Foreman, R. K., Labosky, P. A., Mallo, M. BMP signaling is essential for development of skeletogenic and neurogenic cranial neural crest. Development. 127 (5), 1095-1104 (2000).
  45. Garcia-Lopez, R., Pombero, A., Martinez, S. Fate map of the chick embryo neural tube. Dev. Growth Differ. 51 (3), 145-165 (2009).
  46. Goldstein, A. M., Nagy, N. A bird’s eye view of enteric nervous system development: lessons from the avian embryo. Pediatr. Res. 64 (4), 326-333 (2008).
  47. Le Douarin, N., Dieterlen-Lievre, F., Creuzet, S., Teillet, M. A. Quail-chick transplantations. Methods Cell. Biol. 87, 19-58 (2008).
  48. Wingate, R. J., Lumsden, A. Persistence of rhombomeric organisation in the postsegmental hindbrain. Development. 122 (7), 2143-2152 (1996).
  49. Karagenc, L., Sandikci, M. Tissue distribution of cells derived from the area opaca in heterospecific quail-chick blastodermal chimeras. J. Anat. 216 (1), 16-22 (2010).
  50. Teague, W. J., Jayanthi, N. V., Lear, P. V., Johnson, P. R. Foregut mesenchyme contributes cells to pancreatic acini during embryonic development in a chick-quail chimera model. Pediatr. Surg. Int. 21 (3), 138-142 (2005).
  51. Borue, X., Noden, D. M. Normal and aberrant craniofacial myogenesis by grafted trunk somitic and segmental plate mesoderm. Development. 131 (16), 3967-3980 (2004).
  52. He, L. Three different fates of cells migrating from somites into the limb bud. Anat. Embryol. (Berl). 207 (1), 29-34 (2003).
  53. Huang, R., Zhi, Q., Christ, B. The relationship between limb muscle and endothelial cells migrating from single somite. Anat. Embryol. (Berl). 206 (4), 283-289 (2003).
  54. Hidalgo-Sanchez, M., Simeone, A., Alvarado-Mallart, R. M. Fgf8 and Gbx2 induction concomitant with Otx2 repression is correlated with midbrain-hindbrain fate of caudal prosencephalon. Development. 126 (14), 3191-3203 (1999).
  55. Verberne, M. E., Gittenberger-de Groot, A. C., Poelmann, R. E. Lineage and development of the parasympathetic nervous system of the embryonic chick heart. Anat. Embryol. (Berl). 198 (3), 171-184 (1998).
  56. Burns, A. J., Douarin, N. M. The sacral neural crest contributes neurons and glia to the post-umbilical gut: spatiotemporal analysis of the development of the enteric nervous system. Development. 125 (21), 4335-4347 (1998).
  57. Debby-Brafman, A., Burstyn-Cohen, T., Klar, A., Kalcheim, C. F-Spondin, expressed in somite regions avoided by neural crest cells, mediates inhibition of distinct somite domains to neural crest migration. Neuron. 22 (3), 475-488 (1999).
  58. Lwigale, P. Y., Bronner-Fraser, M. Lens-derived Semaphorin3A regulates sensory innervation of the cornea. Dev. Biol. 306 (2), 750-759 (2007).
  59. Nakamura, H., Funahashi, J. Introduction of DNA into chick embryos by in ovo electroporation. Methods. 24 (1), 43-48 (2001).
  60. Chen, Y. X., Krull, C. E., Reneker, L. W. Targeted gene expression in the chicken eye by in ovo electroporation. Mol. Vis. 10, 874-883 (2004).
  61. Sato, F., Nakagawa, T., Ito, M., Kitagawa, Y., Hattori, M. A. Application of RNA interference to chicken embryos using small interfering RNA. J. Exp. Zool. A. Comp. Exp. Biol. 301 (10), 820-827 (2004).
  62. Lwigale, P. Y., Schneider, R. A. Other chimeras: quail-duck and mouse-chick. Methods Cell. Biol. 87, 59-74 (2008).
  63. Hamburger, V., Hamilton, H. L. A series of normal stages in the development of the chick embryo. 1951. Dev. Dyn. 195 (4), 231-272 (1992).
  64. Brady, J. A simple technique for making very fine, durable dissecting needles by sharpening tungsten wire electrolytically. Bull. World Health Organ. 32 (1), 143-144 (1965).
  65. Le Douarin, N. M. A Feulgen-positive nucleolus. Exp. Cell. Res. 77 (1), 459-468 (1973).
  66. Feulgen, R., Rossenbeck, H. Mikroskopisch-chemischer Nachweis einer Nucleinsaure vom typus der Thymonucleinsiiure und die darauf beruhende elektive Faibung von Zellkemen in mikroskopischen Praparaten. Hoppe-Seyler’s Z. Physiol. Chem. 135, 203-252 (1924).
  67. Lwigale, P. Y., Conrad, G. W., Bronner-Fraser, M. Graded potential of neural crest to form cornea, sensory neurons and cartilage along the rostrocaudal axis. Development. 131 (9), 1979-1991 (2004).
  68. Weston, J. A. A radioautographic analysis of the migration and localization of trunk neural crest cells in the chick. Dev. Biol. 6, 279-310 (1963).
  69. Le Douarin, N. M., Kalcheim, C. . The Neural Crest. , (2009).
  70. Le Douarin, N. M., Teillet, M. A. The migration of neural crest cells to the wall of the digestive tract in avian embryo. J. Embryol. Exp. Morphol. 30 (1), 31-48 (1973).
  71. Douarin, N. M. L. e., Jotereau, F. V. Tracing of cells of the avian thymus through embryonic life in interspecific chimeras. J. Exp. Med. 142 (1), 17-40 (1975).
  72. Le Douarin, N. M., Renaud, D., Teillet, M. A., Le Douarin, G. H. Cholinergic differentiation of presumptive adrenergic neuroblasts in interspecific chimeras after heterotopic transplantations. Proc. Natl. Acad. Sci. U. S. A. 72 (2), 728-732 (1975).
  73. Houssaint, E., Belo, M., Le Douarin, N. M. Investigations on cell lineage and tissue interactions in the developing bursa of Fabricius through interspecific chimeras. Dev. Biol. 53 (2), 250-264 (1976).
  74. Le Douarin, N. M., Jotereau, F. V., Houssaint, E., Belo, M. Ontogeny of the avian thymus and bursa of Fabricius studied in interspecific chimeras. Ann. Immunol. (Paris). 127 (6), 849-856 (1976).
  75. Fontaine, J., Le Douarin, N. M. Analysis of endoderm formation in the avian blastoderm by the use of quail-chick chimaeras. The problem of the neurectodermal origin of the cells of the APUD series. J. Embryol. Exp. Morphol. 41, 209-222 (1977).
  76. Narayanan, C. H., Narayanan, Y. On the origin of the ciliary ganglion in birds studied by the method of interspecific transplantation of embryonic brain regions between quail and chick. J. Embryol. Exp. Morphol. 47, 137-148 (1978).
  77. Lwigale, P. Y., Cressy, P. A., Bronner-Fraser, M. Corneal keratocytes retain neural crest progenitor cell properties. Dev. Biol. 288 (1), 284-293 (2005).
  78. Lwigale, P. Y. Embryonic origin of avian corneal sensory nerves. Dev. Biol. 239 (2), 323-337 (2001).
  79. Tanaka, H., Kinutani, M., Agata, A., Takashima, Y., Obata, K. Pathfinding during spinal tract formation in the chick-quail chimera analysed by species-specific monoclonal antibodies. Development. 110 (2), 565-571 (1990).

Tags

Neural Crest MigrationDifferentiationCross-species TransplantationAvian EmbryosQuail-chick ChimerasMigratory Neural Crest CellsEpithelial To Mesenchymal TransitionCell TypesCartilageMelanocytesNeuronsGliaMultipotent CellsRostro-caudal AxisNeighboring CellsMicroenvironmentPatterningOrganogenesis
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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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