Congenital forms of sensorineural hearing loss can arise from perturbations in development of either peripheral- or central-nervous-system components. Gene discovery approaches that can identify new genes expressed during development of the auditory or vestibular systems in animal models should assist in revealing genetic causes of congenital deafness in humans. We have devised a new Gal4-UAS-based gene- trap screen for zebrafish embryos that should facilitate not only gene discovery, but also both loss-of-function and gain-of-function approaches for testing candidate genes involved in complex processes such as development of the auditory and vestibular systems. Furthermore, our gene-trapping strategy should be applicable to any developing organ or system in the zebrafish, thereby enhancing the versatility of zebrafish as an important model organism for understanding the genetic causes of various human birth defects. We propose two Specific Aims. (1) To generate new otic- or neural-specific gene-trap lines in zebrafish. Pseudotyped retroviral vectors or Tol2 transposases will be used to insert a gene-trap construct into the zebrafish germline. The trapping construct will use a GAL4-UAS system to transactivate the expression of a reporter gene that can be screened by fluorescence imaging of live embryos. Lines showing relatively specific expression in peripheral or central components of mechanosensory systems will be created and trapped genes will be cloned. (2) To use gene-trapped Gal4-driver lines for targeted cell ablation in vivo. One major advantage of our gene-trap design is its potential for targeting bioactive molecules to specific cells in vivo without requiring the isolation of cell- or tissue-specific promoters. This can be accomplished by crossing a particular Gal4-trap line (i.e., the activator line) with a transgenic line carrying a target gene placed downstream of a UAS sequence (i.e., the effector line). Only when both the activator and effector are active in the same cells is the effector protein expressed. An inducible form of Gal4 (GeneSwitch) will permit even more control over the onset of effector protein expression. As proof-of-principle, an effector line will be created with UAS upstream of a toxin gene. When crossed to any of the driver lines, we expect the toxin will specifically kill only those cells expressing the trapped gene. This should prove especially powerful for selective ablation of subsets of CNS neurons to assess their role in development and/or in behavior. Our novel gene-trap screen should facilitate gene discovery in zebrafish, and readily allow tests of candidate genes for their involvement in development of the auditory system. Our long-term goal is to determine whether any of the newly discovered hearing-related genes in zebrafish also correspond to genes underlying congenital deafness in humans. ? ? ?
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