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. We have performed follow up studies on the response of the Stat3/Socs3 pathway during hair cell regeneration and shown that inhibition of Stat3 signaling leads to an increase in hair cell production, accelerating hearing repair. We are currently working on a number of projects to elucidate the role of stat3 in regeneration and to systematically test other candidate genes for their role in hair cell regeneration. To define the role of stat3 during tissue repair/regeneration, we are constructing transgenic zebrafish lines that express a stat3-gfp fusion. This will allow us to perform a variety of studies on the function of stat3 in vivo. A stat3-gfp fusion driven by the endogenous stat3 promoter (cloned as a BAC construct) will monitor both stat3 expression detected by GFP fluorescence, and stat3 activation, shown by the translocation of stat3 protein from the inner cell membrane surface to the nucleus. We will perform time-lapse experiments visualizing the cells involved in lateral line hair cell regeneration, and watching the dynamics of stat3 activation in the presumptive stem cells. In addition, we can observe the change in cell dynamics in the presence of stat3 inhibitors or when cells are expressing a dominant active form of stat3. 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 will recover 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 Initial pilot experiments have shown that 4 mutations out of 40 tested have detectable embyronic phenotypes that affect ear development. This is an approximately ten-fold enrichment over screening of genes at random. We will screen a minimum of 500 mutations, potentially yielding 50 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.

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1
Fiscal Year
2012
Total Cost
$389,441
Indirect Cost
Name
National Human Genome Research Institute
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Vrljicak, Pavle; Tao, Shijie; Varshney, Gaurav K et al. (2016) Genome-Wide Analysis of Transposon and Retroviral Insertions Reveals Preferential Integrations in Regions of DNA Flexibility. G3 (Bethesda) 6:805-17
Pei, Wuhong; Xu, Lisha; Varshney, Gaurav K et al. (2016) Additive reductions in zebrafish PRPS1 activity result in a spectrum of deficiencies modeling several human PRPS1-associated diseases. Sci Rep 6:29946
Chandler, Randy J; LaFave, Matthew C; Varshney, Gaurav K et al. (2016) Genotoxicity in Mice Following AAV Gene Delivery: A Safety Concern for Human Gene Therapy? Mol Ther 24:198-201
Pei, Wuhong; Huang, Sunny C; Xu, Lisha et al. (2016) Loss of Mgat5a-mediated N-glycosylation stimulates regeneration in zebrafish. Cell Regen (Lond) 5:3
Pei, Wuhong; Tanaka, Katsuya; Huang, Sunny C et al. (2016) Extracellular HSP60 triggers tissue regeneration and wound healing by regulating inflammation and cell proliferation. NPJ Regen Med 1:
Planchart, Antonio; Mattingly, Carolyn J; Allen, David et al. (2016) Advancing toxicology research using in vivo high throughput toxicology with small fish models. ALTEX 33:435-452
Gallardo, Viviana E; Varshney, Gaurav K; Lee, Minnkyong et al. (2015) Phenotype-driven chemical screening in zebrafish for compounds that inhibit collective cell migration identifies multiple pathways potentially involved in metastatic invasion. Dis Model Mech 8:565-76
Marquart, Gregory D; Tabor, Kathryn M; Brown, Mary et al. (2015) A 3D Searchable Database of Transgenic Zebrafish Gal4 and Cre Lines for Functional Neuroanatomy Studies. Front Neural Circuits 9:78
Varshney, Gaurav K; Sood, Raman; Burgess, Shawn M (2015) Understanding and Editing the Zebrafish Genome. Adv Genet 92:1-52
Varshney, Gaurav K; Pei, Wuhong; LaFave, Matthew C et al. (2015) High-throughput gene targeting and phenotyping in zebrafish using CRISPR/Cas9. Genome Res 25:1030-42

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