The long-term goal of this research is to understand how cells are specified to form the ear and how these processes are affected in human hearing diseases. The inner ear arises from the otic placode that forms at the lateral edge of the neural plate adjacent to the hindbrain. Current theories suggest that inductive signals from neighboring tissues are specify formation of the placode. However, it is unknown how cells are allocated to the placode or how they respond to the inductive signals that trigger their differentiation into the ear. Previous studies identified and analyzed two sets of transcription factor pairs, DIx3b/4b and Sox9a/9b that interact in a genetic pathway to specify otic placode cells. This pathway is regulated by Fgf3/8 signals from the adjacent hindbrain. The proposed studies will test the hypothesis that DIx3b/4b function is required for cells to become competent to respond to Fgf3/8 inductive signaling and that Fgf3/8 directs convergence and epithelialization of otic precursor cells. ? ? The proposed studies will test whether DIx3b/4b is sufficient for otic competence by examining whether ectopic Fgf3/8 induces placodes only where DIx3b/4b is expressed and whether Fgf3/8 beads induce otic markers in cells that normally Inever contribute to the if cells forced to DIx3b/4b. To learn how DIx3b/4b is ear, these ectopic are express expression regulated by factors that control dorsoventral patterning; functions of Bmps, Chordin and their downstream targets will be altered. These experiments will provide a mechanistic understanding of how cells become competent to form the ear. ? ? The proposed studies will test whether Fgf3/8 directs the morphogenetic movements and epithelialization of otic precursor cell. They will test whether Fgf3/8 is required for these processes by examining embryos in which Fgf3/8 function is blocked by mutation and morpholino treatment. They will test whether Fgf3/8 is sufficient by learning whether Fgf3/8 beads induce ectopic convergence and/or epithelialization. These experiments will elucidate the link between induction and cellular morphogenesis and provide new insights into how cells are specified to form the ear. ? ? A genetic screen of mutant phenotypes will identify additional genes required for induction of the otic placode. These mutations will be characterized phenotypically with mosaic analyses and gene expression analyses in whole-mount embryos and with microarrays to learn how each gene functions, when function is critical and in which cells function is required. The mutations will be characterized genetically by mapping, by complementation testing with existing mutations, and by molecular cloning. This analysis will identify, on the basis of their functions, the critical genes required for formation of the otic placodes. ? ? ?
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