Clusterin has previously been identified as a gene potentially involved in development of the cochlear sensory epithelium. In order to be able to predict the cellular roles that clusterin may play in the development of this organ, an understanding of the spatiotemporal expression pattern is required. Therefore, clusterin gene expression during mouse inner ear development was studied using riboprobes from the mouse gene. Clusterin mRNA demonstrates a dynamic expression pattern within the developing cochlear sensory epithelium. Clusterin mRNA expression is initiated at 12.5dpc (days post coitum) along the entire length of the cochlear sensory epithelium. Throughout in utero development, expression is maintained but becomes progressively restricted in this sensory epithelium. Postnataly expression resolves to specific cellular regions, but clusterin expression ceases at some time point between P2-P17. The analysis of clusterin protein expression revealed this was not restricted to the developing sensory epithelium alone, but also was detected transiently in the periotic mesenchye, otic capsule, as well as Reissner's membrane, a non-sensory epithelium. The detailed localisation of clusterin was compared to other inner ear markers. Using α and β tectorin mRNA markers, clusterin mRNA was shown to localise to the developing inner and outer sulcus and spiral prominence. Clusterin expression also overlaps with both Myosin VIIA and Prox1, markers for hair cells and supporting cells respectively. Clusterin mRNA and protein was absent from the developing mouse vestibular system. In order to study the regulatory mechanisms underlying inner ear clusterin expression, a clusterin BAC was modified by insertion of ZsGreen reporter gene into clusterin genomic regions using recombineering technique. The pronuclear injection of this construct has not been successful so far although these studies are ongoing. Finally in order to determine the fate of clusterin expressing cells after the expression is ceased in the inner ear, clusterin BAC was modified by insertion of Cre recombinant gene at the same location as ZsGreen gene for the generation of Cre transgenic mouse. This transgenic mouse will be crossed with a silent reporter mouse for future clusterin fate mapping studies.

Inner hair cells (IHCs) are the primary sensory receptors of the mammalian cochlea. I employed the whole-cell patch-clamp technique to study voltage responses and ionic currents of IHCs in mice bearing mutations in hair bundle proteins. These mutations, all associated with Usher syndrome, lead to structural and functional defects of the mechanosensory hair bundle. I observed developmental failure in the electrical properties of IHCs from these mutants: a continuation of neonatal spiking instead of the graded receptor potentials seen in control adult IHCs. Voltage-clamp recordings revealed the main cause as the absence of the adult fast potassium (IK,f) current. Outer hair cells (OHCs) are required to amplify the travelling wave to be detected by the IHCs. Optical and whole-cell patch clamp techniques in these same mutants were employed to investigate the development of adult OHCs. I observed a developmental failure in the electrical properties of these OHCs, seen by an absence of the potassium current IK,n. Electromotility and the associated non-linear capacitance were however observed, indicating that prestin is expressed in the mutants. Acid sensitive ion channels (ASICs) have recently been found to be present within the organ of Corti. Here I present data showing the presence of an acid sensitive ion current in both IHCs and OHCs. ASIC1b knockout mice show a response to changes in the extracellular pH suggesting that the current may be carried through a different channel subtype or that compensatory changes occur. The electrical properties of the IHCs develop to maturity in these mice, however the OHCs appear to remain functionally immature displaying a lack of expression of the IK,n current and electromotily. This lack of electromotile function suggests that ASIC1b may be required either for the function of prestins electromotility or for the targeting of prestin to the cell membrane.