Fig. S4

Side effects caused by strong early expression of Gal4/UAS-based expression systems. a) Strong expression of non-inducible Gal4/UAS system in true transgenic fish at early developmental stages (less than 24 hpf) caused serious abnormalities that became evident later in development. d-f) Mifepristone-induced Gal4/UAS expression caused less pronounced developmental abnormalities compared to the non-inducible system when induced before 24hpf. In some transgenic fish the phenotypes were subtle (g) and some survived to maturity. Induction after 24hpf did not cause serious effects although sometimes affected individual cell morphology. For both non-inducible and inducible Gal4/UAS-based systems severity of the phenotypes strongly correlated with the intensity of reporter expression.
a) Developmental abnormalities in transgenic F₁ fish carrying a genomic insertion of the non-inducible EF-GVP-UG cassette (Koster and Fraser, 2001), which included 150-bp Xenopus EF-1α-promoter driving Gal4-VP16 and the EGFP ORF under control of the 14-mer of UAS Gal4-binding sites fused to the fish basal promoter E1b. The cassette was cloned into the Tol2 transposable element (Kawakami et al., 2000) to enhance the transgenesis. Transgenic F₁ offspring from 8 positive founders harboring independent Tol2 insertions was produced and analyzed, all showing very strong EGFP expression and developmental abnormalities. The extent of the abnormalities correlated with the intensity of EGFP expression. In the weakest expressing line the abnormalities became visible only at 3 dpf, however none of the fish survived to adulthood.
b-f) Treatment with 1 μM mifepristone in the growth water 10 hpf onwards.
b) Control wild type fish. c) Transgenic F₂ embryos harboring the LexPR driver-reporter cassette described in this manuscript. Expression of LexA transactivator does not affect embryo development and morphology of the EGFP-positive cells.
d-g) Transgenic F₂ fish (line SWD-G4) carrying the Gal4/UAS-based mifepristone-inducible system described by (Burcin et al., 1999). The cassette harbored a gene for chimeric transcription factor consisting of Gal4 DNA binding domain, a truncated ligand binding domain from the human progesterone receptor and an activation domain of the human NF-kB protein under control of the 0.5 krt8 promoter (identical to the promoter used in LexPR system described in this manuscript); and EGFP gene under control of 6xGal4 upstream activating sequences fused to the minimal adenovirus E1b promoter. The cassette was packed into the Ds transposon vector to induce effective transgenesis. Transgenic embryos were treated with 1 μM mifepristone from 10 hpf onwards. d) 24 hpf, retarded body growth, axis curvature, abnormal yolk extension. e) 48 hpf, underdeveloped brain, branchial arches and heart. f) 5 days, pericardial edema, underdeveloped branchial arches, small brain – a typical phenotype developed in transgenic F₂ fish (SWD-G4 line) following the induction with 1 μM mifepristone at 10 hpf onwards. g) Transgenic F₂ fish (SWD-G4 line), which was induced early but managed showed weak phenotype. Less than 10% of SWD-G4 larvae recovered after 10hpf induction.
Transgenic fish harboring Gal4/UAS-based mifepristone-inducible system exhibited developmental phenotypes only upon induction, suggesting that inducible Gal4-based transactivator require mifepristone binding to cause side effects.