Spatial and Temporal Control of Transgene Expression in Zebrafish

Citation: Akerberg AA, Stewart S, Stankunas K ( Spatial and Temporal Control of Transgene Expression in Zebrafish Alexander A. Akerberg 0 Scott Stewart 0 Kryn Stankunas 0 John F. Rawls, University of North Carolina at Chapel Hill, United States of America 0 1 Institute of Molecular Biology, University of Oregon , Eugene, Oregon , United States of America, 2 Department of Biology, University of Oregon , Eugene, Oregon , United States of America Transgenic zebrafish research has provided valuable insights into gene functions and cell behaviors directing vertebrate development, physiology, and disease models. Most approaches use constitutive transgene expression and therefore do not provide control over the timing or levels of transgene induction. We describe an inducible gene expression system that uses new tissue-specific zebrafish transgenic lines that express the Gal4 transcription factor fused to the estrogen-binding domain of the human estrogen receptor. We show these Gal4-ERT driver lines confer rapid, tissue-specific induction of UAScontrolled transgenes following tamoxifen exposure in both embryos and adult fish. We demonstrate how this technology can be used to define developmental windows of gene function by spatiotemporal-controlled expression of constitutively active Notch1 in embryos. Given the array of existing UAS lines, the modular nature of this system will enable many previously intractable zebrafish experiments. - Funding: Financial support came from the Oregon Medical Research Foundation (SS), a NIH/NICHD Developmental Biology Training Grant (AAA), the University of Oregon (KS), and the NIH/NHLBI (5R00HL087598) (KS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Research using transgenic zebrafish lines has greatly contributed to our understanding of vertebrate biology. Transgenic zebrafish are used widely for both gain and loss of function experiments as well as a means to track specific cell populations. All such studies require careful consideration regarding the location, timing, and levels of transgene expression. For instance, although constitutive ubiquitous promoters generally produce high levels of transgene expression and generate robust phenotypes, they do not differentiate cell-type specificity or the timing of gene function. To overcome these limitations, tissue specific promoters are used to direct transgene expression to discrete cell lineages and tissue types. Temporal control of transgene expression further allows the determination of windows during which a gene functions, a feature particularly useful for developmental biology research. Additionally, multiple roles for a gene during development can be distinguished by timing the induction of an appropriate transgene. Temporal control of transgene expression in zebrafish is typically achieved using heat-shock sensitive promoters [1], although small molecule-controlled inducible promoters also can control the timing and tune levels of transgene expression [2]. One elegant method for transgene expression in metazoans uses the Gal4/UAS two component transcriptional activation switch from Saccharomyces cerevisiae [3,4]. Gal4 is a transcription factor that functions in yeast galactose metabolism [5] and binds to a unique DNA sequence found between the GAL1 and GAL10 genes [6]. A version of this sequence is now commonly referred to as a UAS (for Upstream Activating Sequence). A discrete DNA-binding domain (DBD) of Gal4 is both necessary and sufficient for highspecificity binding to a UAS [7]. Gal4 can activate transcription in heterologous systems by using synthetic promoters consisting of basal transcription initiating elements combined with tandem repeats of the UAS sequence [3,4,810]. Modified Gal4-based chimeric proteins comprising the Gal4 DBD fused to the strong transcriptional activation domain from the viral VP16 protein are particularly potent and specific transcriptional activators [11,12]. The Gal4/UAS system can be readily adaptable for transgenesis studies in diverse biological systems given that any gene of interest can be inserted downstream of a UAS/minimal promoter cassette. In the Gal4/UAS system, the spatial domain of UAS-controlled transgene expression is determined by the promoter used to express Gal4 [13]. Additional refinements of the Gal4/UAS system provide temporal control of transgene expression. Taking advantage of the modular nature of Gal4 [7] and steroid hormone receptors [14], chimeric proteins have been produced that fuse the hormone binding domain from either the estrogen (ER) or glucocorticoid receptor (GR) to Gal4 [14]. The resulting Gal4-ER and Gal4-GR fusion proteins activate UAS-controlled reporter genes only in the presence of the cognate steroid hormone [11,14]. To overcome effects of endogenous steroid hormones on Gal4-ER chimeras, an ER variant (known as ERT) with reduced affinity for naturally occurring estradiol but very high affinity for the estradiol analogs tamoxifen and 4-hydroxy tamoxifen (4-OHT) has been developed [15,16]. The collective features of Gal4-ERT-VP16 fusion proteins make them a potent, highly specific, and tightly controllable tool for transgene expression upon administration of either tamoxifen or 4-OHT [11,12,1416]. The Gal4/UAS system has been adapted for use in zebrafish [1719] and a wide-range of UAS lines are available that express, for example, wild-type or mutant proteins, fluorescent markers, and Cre recombinase for lineage-tracing studies. However, a search of available zebrafish lines from the Zebrafish International Resource Center (ZIRC) yields only a handful of Gal4 lines that use unique, well-characterized promoters. Further, the majority of available Gal4 lines lack the ability to control the timing of gene expression. This is particularly problematic for larval or adult studies when phenotypes arising from earlier transgene expression obscure or exclude the later studies. Inducible Gal4/UAS switches in zebrafish designed to overcome this problem rely on heat shock promoter control of Gal4 expression [1]. This approach produces strong expression but suffers from several major disadvantages, including a lack of cell-type specificity, poor control of expression kinetics, and spurious effects of repeated heat shocks. To overcome these limitations, we adapted the tamoxifen controlled Gal4-ERT system for use in zebrafish. We describe the generation and characterization of three tissue specific tamoxifendependent Gal4-ERT driver lines. These lines achieve rapid, dosedependent expression of UAS-controlled transgenes by simply adding tamoxifen or 4-OHT to zebrafish water. We also validate this approach for functional analysis by demonstrating that tamoxifen dependent, tissue-specific expression of (...truncated)