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. We exposed zebrafish to sound at sufficient decibels to cause significant, measurable hearing damage. We then measured the transcriptional changes in gene expression that occurred over the recovery period of four days and defined the mRNA genetic network needed for hair cell regeneration. We have identified over 2000 genes that are differentially regulated during hair cell regeneration, and we are in the process of systematically testing genes by gene inactivation and phenotyping of the mutant alleles 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. The other major project of the lab Zebrafish insertional mutagenesis and functional genomics, is generating thousands of mutations in the zebrafish genome and we will use mutants isolated from this work to identify other genes critical to proper hair cell regeneration. We have identified over 3,700 mutations by retroviral integration and approximately 200 of those genes are in our candidate lists from the sound exposure experiments. We recovered predicted mutations using in vitro fertilization and raise the mutant carriers to sexual maturity. We will then inbreed sibling carriers and screen 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 8 mutations out of 150 tested have detectable embyronic phenotypes that affect ear development. This is an approximately ten-fold enrichment over screening of genes at random. We have also identified two genes which have an impact on hair cell regeneration without having a significant impact on the normal development of the hair cells. We have now shifted our mutagenesis efforts to CRISPR/Cas9 and because this approach is more efficient than retroviral integration, now our goal is to screen 2000 mutations, potentially yielding 100 new genes involved in hair cell development or regeneration. These genes will be prioritized based on the nature of the phenotypes, with highest priority going to genes that specifically impact hair cell regeneration. All mutations of interest will be freely distributed among the relevant research community for further study. A key gene we are currently actively studying is HSP60. Additionally we are performing pharmacological inhibition of candidate signaling pathways to determine which signals are involved in activation of neuromast hair cell regeneration after ototoxic exposure to copper. Recent experiments in the lab have shown that depending on which TGF-B receptor is inhibited, you can either prevent all hair cell regeneration after ablation in the case of inhibiting ALK3, or you can actually prevent hair cell death completely if you inhibit ALK5. In the next year we will determine which TGF-B ligands are involved in these processes and from which cells in the lateral line neuromasts are these signals coming from. We have recently shown the FDA approved drug Losartan can also act as an otoprotectant in zebrafish against both copper and cisplatin exposure.
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