Neural crest cells go through an epithelial-mesenchymal transition (EMT) before they migrate. B1 integrins are necessary during these phases of neural crest development, but it is unclear if integrins are required for both EMT and neural crest migration. Chimeric integrin B1 subunit and mutant Rho GTPases are used in this study to assess function during neural crest emigration. Cultures of chick embryonic cells, transfected with these constructs, are used to confirm the effects and expression in conjunction with a green fluorescent protein (GFP) reporter. In control experiments targeting the neural ectoderm of the hindbrain by localized electroporation. GFP-expressing cells release from the neural tube, migrate along neural crest pathways and express the HNK-1 neural crest marker. Immunolabeling of Sox9 and Slug neural crest markers shortly after electroporation confirms transfection of prospective neural crest cells. Electroporation with a chimeric hemagglutinin-B1 integrin subunit inhibits release of transfected cells from the neural tube. Embryos electroporated with constitutively active RhoA have a few transfected cells outside the neural tube that express N-cadherin. but they fail to migrate to the branchial arch. Electroporation with constitutively active Rac1 results in numerous cells near the neural tube, none of which express N-cadherin. Embryos electroporated with Cdc42 mutants are not distinguishable from control embryos expressing GFP alone. In embryos co-electroporated with chimeric integrin and dominant negative RhoA together, co-transfected cells migrate along neural crest pathways. The conclusion is that integrin signaling, transduced through RhoA, is necessary for the EMT of cranial neural crest. Key to this investigation of neural crest emigration is the methodology of localized electroporation. This technique introduces transgenes to targeted patches of cells in the embryo. Localized electroporation employs a double-barreled suction electrode to deliver plasmid and produce an electric field. Parameters for localized electroporation are optimized for transfecting a range of cells in the chick embryo, and expansion of the technique to mammals is demonstrated. Localized electroporation has improved reliability and higher efficiency than existing in vivo transfection techniques.
| Identifer | oai:union.ndltd.org:uvic.ca/oai:dspace.library.uvic.ca:1828/2360 |
| Date | 15 March 2010 |
| Creators | Atkins, Ross L. |
| Contributors | Burke, Robert D. |
| Source Sets | University of Victoria |
| Language | English, English |
| Detected Language | English |
| Type | Thesis |
| Rights | Available to the World Wide Web |
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