Immortalized cell lines from embryonic avian and murine otocysts: Tools for molecular studies of the developing inner ear

Abstract

Recently, our studies have focused on genes expressed at the earliest stages of inner ear development. Our aim is to identify and characterize genes that are involved in determining the axes of the semicircular canals, in otic crest delamination and in early innervation of the inner ear. Many elegant studies of auditory development have been done in animal models. However, the need for large amounts of well-characterized embryonic material for molecular studies makes the development of otocyst cell lines with different genetic repertoires attractive. We have therefore derived immortalized otocyst cells from two of the most widely used animal models of ear development: avians and mice. Avian cell isolates were produced from quail otocysts (embryonic stage 19) that were transformed with temperature-sensitive variants of the Rous sarcoma virus (RSV). Among the individual transformed cells are those that produce neuron-like derivatives in response to treatment with 10⁻⁹ M retinoic acid. Mammalian cell isolates were derived from otocysts, of 9 day (post coitus) embryos of the H2k^btsA58 transgennc mouse (Immortomouse), which carries a temperature-sensitive variant of the Simian Virus 40 Tumor antigen. The vast majority of cells of the Immortomouse are capable of being immortalized at 33°C, the permissive temperature for transgene expression, in the presence of γterferon. Several putative clones of these cells differentiated into neuron-like cells after temperature shift and withdrawal of γ-interferon; another isolate of cells assumed a neuron-like morphology on exposure to brain-derived neurotrophic factor even at the permissive temperature. We describe also a cell isolate that expresses the Pax-2 protein product and two putative cell lines that express the protein product of the chicken equivalent of the Drosophila segmentation gene engrailed. These genes and their protein products are expressed in specific subpopulations of otocyst cells at early stages. Both mouse and quail immortalized cell lines will be used to study inner ear development at the molecular level.