A model for mammalian cochlear hair cell differentiation in vitro: effects of retinoic acid on cytoskeletal proteins and potassium conductances.
We have established a model for the in-vitro differentiation of mouse cochlear hair cells and have used it to explore the influence of retinoic acid on proliferation, cytoskeletal proteins and voltage-gated potassium conductances. The model is based on the conditionally immortal cell line University of Sheffield/ventral otocyst-epithelial cell line clone 36 (US/VOT-E36), derived from ventral otic epithelial cells of the mouse at embryonic day 10.5 and transfected with a reporter for myosin VIIa. Retinoic acid did not increase cell proliferation but led to up-regulation of myosin VIIa and formation of prominent actin rings that gave rise to numerous large, linear actin bundles. Cells expressing myosin VIIa had larger potassium conductances and did not express the cyclin-dependent kinase inhibitor p27(kip1). US/VOT-E36 endogenously expressed the voltage-gated potassium channel alpha-subunits Kv1.3 and Kv2.1, which we subsequently identified in embryonic and neonatal hair cells in both auditory and vestibular sensory epithelia in vivo. These subunits could underlie the embryonic and neonatal delayed-rectifiers recorded in nascent hair cells in vivo. Kv2.1 was particularly prominent on the basolateral membrane of cochlear inner hair cells. Kv1.3 was distributed throughout all hair cells but tended to be localized to the cuticular plates. US/VOT-E36 recapitulates a coherent pattern of cell differentiation under the influence of retinoic acid and will provide a convenient model for screening the effects of other extrinsic factors on the differentiation of cochlear epithelial cell types in vitro.
Helyer
R
R
Department of Biomedical Science, Addison Building, Western Bank, Sheffield, UK.
Cacciabue-Rivolta
D
D
Davies
D
D
Rivolta
M N
MN
Kros
C J
CJ
Holley
M C
MC
D000818:Animals; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins / Q000037:antagonists & inhibitors / Q000494:pharmacology; D002452:Cell Count; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D003051:Cochlea / Q000166:cytology*; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D004347:Drug Interactions; D004622:Embryo, Mammalian; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D023421:Models, Animal; D011188:Potassium / Q000378:metabolism*; D024642:Potassium Channels, Voltage-Gated / Q000378:metabolism; D014162:Transfection / Q000379:methods; D014212:Tretinoin / Q000494:pharmacology*
C525810:Bmp4 protein, mouse; D055415:Bone Morphogenetic Protein 4; D019485:Bone Morphogenetic Proteins; D016895:Culture Media, Serum-Free; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; D024642:Potassium Channels, Voltage-Gated; D049452:Green Fluorescent Proteins; D014212:Tretinoin; D011188:Potassium