Abstract
The vertebrate inner ear is composed of several specialized epithelia containing mechanosensory “hair” cells, sensitive to sound and head movements. In mammals, the loss of hair cells for example during aging or after noise trauma is irreversible and results in permanent sensory deficits. By contrast, avian, fish, and amphibians can efficiently regenerate lost hair cells following trauma. The chicken inner ear is a classic model system to investigate the cellular and molecular mechanisms of inner ear development and regeneration, yet it suffered until recently from a relative lack of flexible tools for genetic studies. With the introduction of in ovo electroporation and of Tol2 transposon vectors for gene transfer in avian cells, the field of experimental possibilities has now expanded significantly in this model. Here we provide a general protocol for in ovo electroporation of the chicken otic placode and illustrate how this approach, combined with Tol2 vectors, can be used to drive long-term and inducible gene expression in the embryonic chicken inner ear. This method will be particularly useful to investigate the function of candidate genes regulating progenitor cell behavior and sensory cell differentiation in the inner ear.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fekete DM, Wu DK (2002) Revisiting cell fate specification in the inner ear. Curr Opin Neurobiol 12:35–42
Bok J, Chang W, Wu DK (2007) Patterning and morphogenesis of the vertebrate inner ear. Int J Dev Biol 51:521–533
Stone J, Rubel E (2000) Cellular studies of auditory hair cell regeneration in birds. Proc Natl Acad Sci USA 97:11714–11721
Stone JS, Cotanche DA (2007) Hair cell regeneration in the avian auditory epithelium. Int J Dev Biol 51:633–647
Bok J, Bronner-Fraser M, Wu DK (2005) Role of the hindbrain in dorsoventral but not anteroposterior axial specification of the inner ear. Development 132:2115–2124
Groves A, Bronner-Fraser M (2000) Competence, specification and commitment in otic placode induction. Development 127:3489–3499
Nakamura H, Funahashi J (2001) Introduction of DNA into chick embryos by in ovo electroporation. Methods 24:43–48
Momose T, Tonegawa A, Takeuchi J, Ogawa H, Umesono K, Yasuda K (1999) Efficient targeting of gene expression in chick embryos by microelectroporation. Dev Growth Differ 41:335–344
Alsina B, Abelló G, Ulloa E, Henrique D, Pujades C, Giraldez F (2004) FGF signaling is required for determination of otic neuroblasts in the chick embryo. Dev Biol 267:119–134
Daudet N, Lewis J (2005) Two contrasting roles for Notch activity in chick inner ear development: specification of prosensory patches and lateral inhibition of hair-cell differentiation. Development 132:541–551
Hughes S, Greenhouse J, Petropoulos C, Sutrave P (1987) Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors. J Virol 61:3004–3012
Kiernan A, Fekete D (1997) In vivo gene transfer into the embryonic inner ear using retroviral vectors. Audiol Neurootol 2:12–24
Eddison M, Le Roux I, Lewis J (2000) Notch signaling in the development of the inner ear: lessons from Drosophila. Proc Natl Acad Sci USA 97:11692–11699
Bird JE, Daudet N, Warchol ME, Gale JE (2010) Supporting cells eliminate dying sensory hair cells to maintain epithelial integrity in the avian inner ear. J Neurosci 30:12545–12556
Sato Y, Kasai T, Nakagawa S, Tanabe K, Watanabe T, Kawakami K, Takahashi Y (2007) Stable integration and conditional expression of electroporated transgenes in chicken embryos. Dev Biol 305:616–624
Watanabe T, Saito D, Tanabe K, Suetsugu R, Nakaya Y, Nakagawa S, Takahashi Y (2007) Tet-on inducible system combined with in ovo electroporation dissects multiple roles of genes in somitogenesis of chicken embryos. Dev Biol 305:625–636
Koga A, Iida A, Hori H, Shimada A, Shima A (2006) Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces. Mol Biol Evol 23:1414–1419
Kawakami K (2005) Transposon tools and methods in zebrafish. Dev Dyn 234:244–254
Acknowledgements
N.D. thanks Dr. Alexander Davies for teaching him the basics of in ovo electroporation. This work was supported by Deafness Research UK and the Biotechnology and Biological Sciences Research Council (BBSRC).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media New York
About this protocol
Cite this protocol
Freeman, S., Chrysostomou, E., Kawakami, K., Takahashi, Y., Daudet, N. (2012). Tol2-Mediated Gene Transfer and In Ovo Electroporation of the Otic Placode: A Powerful and Versatile Approach for Investigating Embryonic Development and Regeneration of the Chicken Inner Ear. In: Mace, K., Braun, K. (eds) Progenitor Cells. Methods in Molecular Biology, vol 916. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-980-8_10
Download citation
DOI: https://doi.org/10.1007/978-1-61779-980-8_10
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-979-2
Online ISBN: 978-1-61779-980-8
eBook Packages: Springer Protocols