Regulatory analysis of Fgf3 and Fgf10 and the role of retinoids in their control

With 9 million people in the UK alone suffering from deafness or hearing impairment, there is a concerted effort to develop effective treatments. To address this, one strategy is to recruit mechanisms naturally occurring during inner ear development, but this first requires a clear picture of the normal molecular mechanisms underlying this developmental process. Fibroblast growth factors (FGFs) are short-range extracellular signalling molecules, with Fgf3 and Fgf10 already shown to be critical for the earliest event of inner ear induction. Interestingly these ligands are also expressed in the inner ear itself, and mutations in both Fgf3 and Fgf10 have independently been linked to sensorineural deafness in humans. This project is focussed on unravelling the molecular mechanisms controlling their expression in the inner ear. Bioinformatic analysis of the Fgf3 and Fgf10 enhancer regions revealed the presences of putative binding sites for retinoic acid (RA). RA is a key signalling molecule in inner ear development with both excess and deficit leading to inner ear abnormalities. First, a novel, non-invasive method of RA administration via sugar pellet was tested and proved to be efficient and reliable alternative to gavage. Using an Fgf3-lacZ reporter mouse, the effects of RA excess on Fgf3 expression were investigated in detail. In addition, preliminary studies of the effects of RA on Fgf10 were also carried out. Both Fgf3 and Fgf10 were downregulated with high doses of RA, confirming previous in vitro studies. In addition, detailed analysis of Fgf3-lacZ embryos exposed to RA revealed that downregulation critically depends on the dose and time of administration. To further explore other direct regulators of Fgf10 expression, additional reporter constructs were also generated for functional analysis in mouse and in chick. To date, analysis of electroporated chick embryos shows that ear specific Fgf10 regulation may not be conserved between two species.