Fig. 1

Generation of the GRE-responsive line and developmental profile of GRE-mediated transcriptional activity of Tg(9×GCRE-HSV.Ul23:EGFP)ia20 embryos and larvae (transgenic males crossed with WT females). (A) Schematic representation of the 9×GCRE-HSV.Ul23:EGFP reporter construct designed in this study. The construct consists of a 0.45-kb fragment encoding nine repeating GREs (in black) from the rat tyrosine aminotransferase promoter ( Grange et al., 1991) and a thymidine kinase (tk) promoter (HSV.Ul23, in dark grey) inserted upstream of the egfp ORF (in green). The SV40 polyA signal (SV40pA in grey), which contains a transcriptional termination element, is directly downstream of the EGFP ORF and the entire element is flanked by Tol2 transposable elements. (B) RT-PCR analysis of egfp expression from the offspring of transgenic female or male and wild type mates at stages ranging from 0 up to 24 hpf. Expression of ef1a was used as a cDNA loading control. Fluorescence microscopy (C-F) and light microscopy (C′-F′) lateral views of zebrafish embryos and larvae at 14 hpf and 1, 3 and 6 dpf displaying the EGFP accumulation sites and the egfp mRNA localization during development. The protein and mRNA are first detectable at 14 hpf, when they both are ubiquitously distributed, with higher signals in the developing head and tail region (C-C′). The EGFP protein/mRNA localization remains the same by 1 dpf (D-D′), while it is visible in distinct domains, such as brain, liver and pronephros at 3 dpf (E-E′). By 6 dpf, the reporter expression is particularly intense in liver, intestine and pronephros (F-F′). Scale bar: 200 µM.