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Fig. 4 ERT2-GAL4 under the control of the ubiquitin promoter can drive widespread, 4-OHT-dependent activity to 24 hours post-fertilisation. (A-K) Stable ubi::ERT2-GAL4 lines were crossed to a UAS::H2B-citrine line, and embryos were treated overnight with ethanol (A,D,G) or 0.75 μM 4-OHT (B,C,E,F,H-K) from 50% epiboly to 21 hpf. Confocal analysis of single optical planes reveals near-ubiquitous GAL4 activity in a 4-OHT-dependent manner in one line (A,D,G versus B,C,E,F,H,I, Citrine in green channel, DAPI in red channel). Arrowheads indicate Citrine-negative cells. A second independent ubi::ERT2-GAL4 line showed much weaker and more mosaic expression (J,K versus H,I, Citrine in green channel, DAPI in red channel), suggesting insertion site-dependent effects on ubiquitin promoter activity. Representative Citrine-negative cells in E,H are indicated by arrowheads. (L) Upon withdrawal of 4-OHT from Citrine-positive embryos from 18 hpf to 42 hpf (24 hour withdrawal, washouts A, B and C), mRNA levels of citrine drop by 67-85% compared with embryos maintained in 4-OHT (+4-OHT) (n=3 clutches) (compare +4-OHT with washouts A, B and C). qPCR levels are relative to embryos maintained in 4-OHT. Error bars indicate ±s.e.m. (overall signficant difference between +4-OHT and washout conditions, t-test, P<0.002).