Hearing regeneration in zebrafish

National Human Genome Research Institute

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Abstract

The Developmental Genomics Section has been using a combination of zebrafish genetics and molecular embryology to study ear development and hearing regeneration. We have initiated research on hair cell regeneration in adult zebrafish. Using a transgenic ablation system, we cause measurable hearing damage. We then measured the transcriptional and chromosomal accessibility changes at the single cell level that occurred over the recovery period of four days and defined the genetic network needed for hair cell regeneration. We have identified over 20,000 loci that are differentially regulated during hair cell regeneration, and we are in the process of systematically testing the gene regulatory network through inactivation and phenotyping of the of enhancers for hair cell development and regeneration in early embryos. These experiments will define the nature of the stem cell niche and some of the conditions necessary to activate the regeneration response. We have mutated 50 enhancers so far of those in our candidate lists from the hair cell ablation experiments. Any positive results from "crispant" experiments will then be tested by inbreeding sibling carriers and screening the embryonic offspring using a variety of measures: 1) visual characterization of the morphology of the inner ear and lateral line in the first 5 days of development 2) efficacy of hair cell regeneration in the lateral line at 5 days post-fertilization 3) vigor of homozygous mutants raised to adult 4) efficacy of the startle response in adults 5) developmental and regeneration defects in embryos from homozygous mutant parents To date, our experiments have shown that 4 mutations out of 50 tested have detectable embyronic phenotypes that affect hearing regeneration. From or previous data, this is an approximately ten-fold enrichment over screening genes at random. Our goal is to screen 1000 enhancers, potentially yielding 80 new genes and enhancers involved in hair cell development or regeneration. These loci will be prioritized based on the nature of the phenotypes, with highest priority going to enhancers that specifically impact hair cell regeneration. All mutations of interest will be freely distributed among the relevant research community for further study.