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Manipulation of gene expression is one of the most informative ways to study gene function. Genetic screens have been an informative method to identify genes involved in developmental processes. In the zebrafish, loss-of-function screens have been the primary approach for these studies. We sought to complement loss-of-function screens using an unbiased approach to overexpress genes with a Gal4-UAS based system, similar to the gain-of-function screens in Drosophila. Using MMLV as a mutagenic vector, a cassette containing a UAS promoter was readily inserted in the genome, often at the 5' end of genes, allowing Gal4-dependent overexpression. We confirmed that genes downstream of the viral insertions were overexpressed in a Gal4-VP16 dependent manner. We further demonstrate that misexpression of one such downstream gene gucy2F, a membrane-bound guanylate cyclase, throughout the nervous system results in multiple defects including a loss of forebrain neurons. This suggests proper control of cGMP production is important in neuronal survival. From this study, we propose that this gain-of-function approach can be applied to large-scale genetic screens in a vertebrate model organism and may reveal previously unknown gene function.