The function of the zinc finger transcription factor GATA3 was studied in a newly established, conditionally immortal cell line derived to represent auditory sensory neuroblasts migrating from the mouse otic vesicle at embryonic day E10.5. The cell line, US/VOT-33, expressed GATA3, the bHLH transcription factor NeuroD and the POU-domain transcription factor Brn3a, as do auditory neuroblasts in vivo. When GATA3 was knocked down reversibly with antisense oligonucleotides, NeuroD was reversibly down-regulated. Auditory and vestibular neurons form from neuroblasts that express NeuroD and that migrate from the antero-ventral, otic epithelium at E9.5-10.5. On the medial side, neuroblasts and epithelial cells express GATA3 but on the lateral side they do not. At E13.5 most auditory neurons express GATA3 but no longer express NeuroD, whereas vestibular neurons express NeuroD but not GATA3. Neuroblasts expressing NeuroD and GATA3 were located in the ventral, otic epithelium, the adjacent mesenchyme and the developing auditory ganglion. The results suggest that auditory and vestibular neurons arise from different, otic epithelial domains and that they gain their identity prior to migration. In auditory neuroblasts, NeuroD appears to be dependent on the expression of GATA3.

Mammalian auditory hair cells are few in number, experimentally inaccessible, and do not proliferate postnatally or in vitro. Immortal cell lines with the potential to di¡erentiate into auditory hair cells would substantially facilitate auditory research, drug development, and the isolation of critical molecules involved in hair cell biology. We have established two conditionally immortal cell lines that express at least ¢ve characteristic hair cell markers. These markers are the transcription factor Brn3.1, the a9 subunit of the acetylcholine receptor, the stereociliary protein ¢mbrin and the myosins VI and VIIA. These hair cell precursors permit functional studies of cochlear genes and in the longer term they will provide the means to explore therapeutic methods of stimulating auditory hair cell regeneration.

We provide evidence from a newly established, conditionally immortal cell line (UB/UE-1) that vestibular supporting cells from the mammalian inner ear can differentiate postnatally into more than one variant of hair cell. A clonal supporting cell line was established from pure utricular sensory epithelia of H2k(b)tsA58 transgenic mice 2 d after birth. Cell proliferation was dependent on conditional expression of the immortalizing gene, the "T" antigen from the SV40 virus. Proliferating cells expressed cytokeratins, and patch-clamp recordings revealed that they all expressed small membrane currents with little time-dependence. They stopped dividing within 2 d of being transferred to differentiating conditions, and within a week they formed three defined populations expressing membrane currents characteristic of supporting cells and two kinds of neonatal hair cell. The cells expressed several characteristic features of normal hair cells, including the transcription factor Brn3.1, a functional acetylcholine receptor composed of alpha9 subunits, and the cytoskeletal proteins myosin VI, myosin VIIa, and fimbrin. Immunofluorescence labeling and electron microscopy showed that the cells formed complex cytoskeletal arrays on their upper surfaces with structural features resembling those at the apices of normal hair cells. The cell line UB/UE-1 provides a valuable in vitro preparation in which the expression of numerous structural and physiological components can be initiated or upregulated during early stages of mammalian hair cell commitment and differentiation.

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This review describes the H2k^btsA58 transgenic mouse (Immortomouse) and its application to the production of conditionally immortalised cell lines from sensory epithelia within the mammalian inner ear. Established cell lines should overcome many of the technical difficulties associated with experimental procedures in auditory and vestibular research. These include the limited amount of tissue available and the relatively complex and laborious dissection. Conditional immortalisation should also allow essential studies on the molecular and cellular mechanisms that govern both the differentiation of sensory cells and the development of sensory epithelia.

In this study, we have shown that human tonsillar T cells adhere to phorbol 12-myristate 13-acetate(PMA)-differentiated U-937 cells. To examine the molecular mechanisms involved, the effect of a panel of monoclonal antibodies upon this adhesion was assessed in a quantitative binding assay. Antibodies against LFA-1 and ICAM-1 inhibited binding, directly implicated these molecules in T cell-PMA-induced U-937 adhesion. Furthermore, the adhesion was magnesium but not calcium dependent. Of the remaining antibodies that were tested, none of those against CD2, LFA-3, Mac-1, p150,95, CD43, CD45RA or CD56 affected binding. However, antibodies against CD44, CD45, CD45RO, CD46 and CD55 enhanced binding suggesting an anti-adhesive role for these molecules during U-937-T cell interaction.

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