Fig. 2

Activity of ERT2-GAL4 in vivo is 4-OHT dependent, rapid and dose sensitive. (A-K) Stable transgenic fish lines expressing ERT2-GAL4 under control of the Xenopus EF1α promoter show robust transactivation of UAS-linked H2B-citrine (C,D), GFP-dnCasp9 (G,H) or Myc-hoxb8a (J,K) when treated overnight with 0.5 μM 4-OHT (all in green channels), compared with controls (A,B,E,F,I, green channel). In some samples, nuclei are labelled with DAPI (B,D,I-K, red channel) and/or brightfield (A-H, grey) to visualize the embryo. (L-V) A time-course of UAS-linked transgene transcription (in situ hybridisation for GFP, purple) driven by ERT2-GAL4 shows 4-OHT dependence (controls in N,Q,T versus 4-OHT treated in O,R,U and P,S,V). The kinetics and intensity of response are dose dependent: embryos treated with 5 μM 4-OHT show more rapid, stronger and less mosaic transcription from the UAS-driven transgene than 0.5 μM 4-OHT-treated embryos (compare P,S,V with O,R,U) over the 6-hour treatment course. Embryos treated overnight with 0.5 μM 4-OHT (control in L, 4-OHT in M) show robust GFP transcription. The light pigmentation around the eye of embryos at 6 hours in T is not in situ signal.