Our long term goal is to understand how cells are specified to form the ear. The inner ear arises from the otic placode. Previous studies suggest that inductive signals from neighboring tissues specify formation of the placode. However, it is unknown how cells are allocated to the placode or how they respond to the signals that trigger their differentiation into the ear. We have identified a zebrafish mutant that lacks a detectable otic placode and fails to form an ear. The mutation is a deficiency of the closely linked dlx3 and dlx7 genes. We propose to use this mutation to analyze the cellular interactions required to form the placode and the potential roles of these genes in this process. 1. We will test the hypothesis that function of the dlx3-dlx7 genes is required for formation of the ear. To learn whether expression of dlx3-dlx7 is necessary to form the ear, we will isolate point mutations in these genes. To learn whether dlx3-dlx7 expression is sufficient, we will rescue the mutant phenotype with wild-type transgenes. These experiments will elucidate the functions of dlx3-dlx7 in specification of otic placode cells. 2. We will test the hypothesis that the dlx3-dlx7 genes function to specify the competence of cells to form the ear. This hypothesis predicts the mutation should autonomously affect cells that form the placode. We will identify which cells the mutation affects by generating genetic mosaics. This analysis will tell us in which cells the dlx3-dlx7 genes normally act. We will use small groups of competent cells from wild-type hosts as probes to learn the locations of signaling cells in mutant hosts. We will then transplant wild-type cells into these locations in dlx3-dlx7 mutants at various developmental stages. These experiments will reveal which cells signal induction of the placode and at what developmental time the induction occurs. 3. We will screen for mutations in genes that encode the inductive signal and signal response system that are required to form the otic placode. We will screen for mutations that block formation of the otic placodes. We will characterize these mutations phenotypically and genetically. This analysis will identify genes on the basis of their functions in specifying the fates of cells that form the otic placodes. This application is part of an IRPG to understand the mechanisms that regulate morphogenesis and cell specification in the vertebrate inner ear.
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