Spatial and temporal control of transgene expression in zebrafish

Akerberg et al. (2014). PLoS ONE DOI: 10.1371/journal.pone.0092217 Citations: 42

Zebrafish induction used heat-shock promoters, lacking cell-type control. This paper built tamoxifen-inducible Gal4-ERT-VP16 lines for tissue-specific activation. EGFP appeared within 3 hours of 4-OHT, and Notch1 lost its notochord effect after 10 hpf.

Parts used

EGFPmCherryEF1α PromoterMyc tag

Key findings

  • Three tissue-specific zebrafish transgenic lines expressing Gal4-ERT-VP16 fusion proteins (driven by krt5, dusp6, and ef1a promoters) achieved tamoxifen-dependent transgene activation with rapid kinetics and dose-dependent expression levels. Treatment with 4-hydroxytamoxifen concentrations from 0.5-2 μM produced detectable UAS-EGFP expression within 3 hours and robust expression by 4.5 hours in embryos. Increasing 4-OHT from 0.5 to 2 μM yielded a 28% increase in transgene levels (P<0.05) without toxic effects.

  • Tamoxifen-inducible expression of constitutively active Notch1 intracellular domain (NICD) in dusp6-positive midline cells revealed a critical developmental window for notochord formation during zebrafish gastrulation. Embryos treated with 4 μM 4-OHT at or before 6 hpf displayed fully penetrant notochord defects (27% affected, matching expected 25% double transgenic frequency), while treatment at 8 hpf showed reduced penetrance and treatment at 10 hpf produced no phenotype despite confirmed NICD expression.

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Abstract

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 UAS-controlled 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.