Abstract
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.
Similar content being viewed by others
References
Amsterdam A, Burgess S, Golling G, Chen W, Sun Z, Townsend K, Farrington S, Haldi M, Hopkins N (1999) A large-scale insertional mutagenesis screen in zebrafish. Genes Dev 13:2713–2724
Asakawa K, Suster ML, Mizusawa K, Nagayoshi S, Kotani T, Urasaki A, Kishimoto Y, Hibi M, Kawakami K (2008) Genetic dissection of neural circuits by Tol2 transposon-mediated Gal4 gene and enhancer trapping in zebrafish. Proc Natl Acad Sci USA 105:1255–1260
Baehr W, Karan S, Maeda T, Luo DG, Li S, Bronson JD, Watt CB, Yau KW, Frederick JM, Palczewski K (2007) The function of guanylate cyclase 1 and guanylate cyclase 2 in rod and cone photoreceptors. J Biol Chem 282:8837–8847
Chen W, Casey Corliss D (2004) Three modules of zebrafish Mind bomb work cooperatively to promote Delta ubiquitination and endocytosis. Dev Biol 267:361–373
Chen W, Burgess S, Hopkins N (2001) Analysis of the zebrafish smoothened mutant reveals conserved and divergent functions of hedgehog activity. Development 128:2385–2396
Chen W, Burgess S, Golling G, Amsterdam A, Hopkins N (2002) High-throughput selection of retrovirus producer cell lines leads to markedly improved efficiency of germ line-transmissible insertions in zebra fish. J Virol 76:2192–2198
Davison JM, Akitake CM, Goll MG, Rhee JM, Gosse N, Baier H, Halpern ME, Leach SD, Parsons MJ (2007) Transactivation from Gal4-VP16 transgenic insertions for tissue-specific cell labeling and ablation in zebrafish. Dev Biol 304:811–824
Desai BS, Monahan AJ, Carvey PM, Hendey B (2007) Blood-brain barrier pathology in Alzheimer’s and Parkinson’s disease: implications for drug therapy. Cell Transplant 16:285–299
Driever W, Solnica-Krezel L, Schier AF, Neuhauss SC, Malicki J, Stemple DL, Stainier DY, Zwartkruis F, Abdelilah S, Rangini Z, Belak J, Boggs C (1996) A genetic screen for mutations affecting embryogenesis in zebrafish. Development 123:37–46
Ellingsen S, Laplante MA, Konig M, Kikuta H, Furmanek T, Hoivik EA, Becker TS (2005) Large-scale enhancer detection in the zebrafish genome. Development 132:3799–3811
Goldsmith P, Harris WA (2003) The zebrafish as a tool for understanding the biology of visual disorders. Semin Cell Dev Biol 14:11–18
Golling G, Amsterdam A, Sun Z, Antonelli M, Maldonado E, Chen W, Burgess S, Haldi M, Artzt K, Farrington S, Lin SY, Nissen RM, Hopkins N (2002) Insertional mutagenesis in zebrafish rapidly identifies genes essential for early vertebrate development. Nat Genet 31:135–140
Grabher C, Joly JS, Wittbrodt J (2004) Highly efficient zebrafish transgenesis mediated by the meganuclease I-SceI. Methods Cell Biol 77:381–401
Haffter P, Nusslein-Volhard C (1996) Large scale genetics in a small vertebrate, the zebrafish. Int J Dev Biol 40:221–227
Hay BA, Maile R, Rubin GM (1997) P element insertion-dependent gene activation in the Drosophila eye. Proc Natl Acad Sci USA 94:5195–5200
Kawakami K, Takeda H, Kawakami N, Kobayashi M, Matsuda N, Mishina M (2004) A transposon-mediated gene trap approach identifies developmentally regulated genes in zebrafish. Dev Cell 7:133–144
Kay JN, Link BA, Baier H (2005) Staggered cell-intrinsic timing of ath5 expression underlies the wave of ganglion cell neurogenesis in the zebrafish retina. Development 132:2573–2585
Kotani T, Nagayoshi S, Urasaki A, Kawakami K (2006) Transposon-mediated gene trapping in zebrafish. Methods 39:199–206
Lin S, Gaiano N, Culp P, Burns JC, Friedmann T, Yee JK, Hopkins N (1994) Integration and germ-line transmission of a pseudotyped retroviral vector in zebrafish. Science 265:666–669
Molnar C, Lopez-Varea A, Hernandez R, de Celis JF (2006) A gain-of-function screen identifying genes required for vein formation in the Drosophila melanogaster wing. Genetics 174:1635–1659
Monahan AJ, Warren M, Carvey PM (2008) Neuroinflammation and peripheral immune infiltration in Parkinson’s disease: an autoimmune hypothesis. Cell Transplant 17:363–372
Muto A, Orger MB, Wehman AM, Smear MC, Kay JN, Page-McCaw PS, Gahtan E, Xiao T, Nevin LM, Gosse NJ, Staub W, Finger-Baier K, Baier H (2005) Forward genetic analysis of visual behavior in zebrafish. PLoS Genet 1:e66
Rorth P (1996) A modular misexpression screen in Drosophila detecting tissue-specific phenotypes. Proc Natl Acad Sci USA 93:12418–12422
Rorth P, Szabo K, Bailey A, Laverty T, Rehm J, Rubin GM, Weigmann K, Milan M, Benes V, Ansorge W, Cohen SM (1998) Systematic gain-of-function genetics in Drosophila. Development 125:1049–1057
Scott EK, Mason L, Arrenberg AB, Ziv L, Gosse NJ, Xiao T, Chi NC, Asakawa K, Kawakami K, Baier H (2007) Targeting neural circuitry in zebrafish using GAL4 enhancer trapping. Nat Methods 4:323–326
Staudt N, Molitor A, Somogyi K, Mata J, Curado S, Eulenberg K, Meise M, Siegmund T, Hader T, Hilfiker A, Bronner G, Ephrussi A, Rorth P, Cohen SM, Fellert S, Chung HR, Piepenburg O, Schafer U, Jackle H, Vorbruggen G (2005) Gain-of-function screen for genes that affect Drosophila muscle pattern formation. PLoS Genet 1:e55
Suenobu N, Shichiri M, Iwashina M, Marumo F, Hirata F (1999) Natriuretic peptides and nitric oxide induce endothelial apoptosis via a cGMP-dependent mechanism. Arterioscler Thromb Vasc Biol 19:140–146
Wang D, Jao LE, Zheng N, Dolan K, Ivey J, Zonies S, Wu X, Wu K, Yang H, Meng Q, Zhu Z, Zhang B, Lin S, Burgess SM (2007) Efficient genome-wide mutagenesis of zebrafish genes by retroviral insertions. Proc Natl Acad Sci USA 104:12428–12433
Wu X, Li Y, Crise B, Burgess SM (2003) Transcription start regions in the human genome are favored targets for MLV integration. Science 300:1749–1751
Yang RB, Garbers DL (1997) Two eye guanylyl cyclases are expressed in the same photoreceptor cells and form homomers in preference to heteromers. J Biol Chem 272:13738–13742
Yang RB, Foster DC, Garbers DL, Fulle HJ (1995) Two membrane forms of guanylyl cyclase found in the eye. Proc Natl Acad Sci USA 92:602–606
Zabel U, Weeger M, La M, Schmidt HH (1998) Human soluble guanylate cyclase: functional expression and revised isoenzyme family. Biochem J 335(Pt 1):51–57
Acknowledgments
We would like to thank Emily Janega for assistance with PCR and In situ hybridization. This work is supported by NIH EY016092 to (WC), NIH Training Grant 1T32HD049309-01A1 to Jan Christian (LAM) and P40 RR012546 (ZIRC).
Author information
Authors and Affiliations
Corresponding author
Additional information
Communicated by T. Becker.
Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. FJ151012, FJ151013, and FJ151014.
Rights and permissions
About this article
Cite this article
Maddison, L.A., Lu, J., Victoroff, T. et al. A gain-of-function screen in zebrafish identifies a guanylate cyclase with a role in neuronal degeneration. Mol Genet Genomics 281, 551–563 (2009). https://doi.org/10.1007/s00438-009-0428-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-009-0428-8