Fig. 2

Fig. 2 Transgenic zebrafish can report on Notch-signaling activity. The Notch-responsive element was used to generate two transgenic lines Tg(Tp1bglob:eGFP)^um14 and Tg(Tp1bglob:hmgb1-mCherry)^jh11. In-crossing homozygous animals for both lines gave clutches expressing fluorescent marker in all individuals. Representatives (at 24hpf) for each lines are shown in (A) and (G). Incubation of clutch mates with the Notch-signaling inhibitor, DAPT (100 μM) from 80% epiboly to 24hpf, results in reduction in fluorescent protein (B and H). This demonstrates that inhibiting Notch-signaling concomitantly reduces Tp1 transgene activity. Over-expression of NICD in our red Notch-responsive line was carried out using a Gal4/UAS bipartite system (Scheer and Campos-Ortega, 1999). (C–E) Schematics of the following constructs: (C) Tp1bglob:hmgb1-mCherry, (D) UAS:notch1a-intra and (E) hsp70l:Gal4. In the presence of all three constructs and following heat-shock, the transcriptional activator Gal4 is expressed and transactivates through its upstream-activating site (UAS) (dashed arrow, E), and this directs expression of NICD. This exogenous NICD augments the endogenous NICD (dashed arrow, D), and in concert with RBP-J_κ and co-factors increases activity of the Tp1 element. (F) Progeny from a Tg(Tp1bglob:hmgb1-mCherry; UAS:notch1a-intra; hsp70l:Gal4) x Tg(UAS:notch1a-intra) cross were selected for red fluorescence and then either heat-shocked or kept as a control. Six hours later embryos were scored for red fluorescence that was enhanced above that seen for controls. All embryos were then genotype for presence of hsp70l:Gal4 (hsp) and UAS:notch1a-intra (UAS) transgenes. As can been seen in (F) and the examples shown in (I-L), enhanced red fluorescence is dependent on heat-shock and possession of both the Gal4 and UAS transgenes (inset panel shows genotyping, UAS/? Refers to either UAS/UAS or UAS/+). Together these results indicate that over-expression of NICD augments expression from the Tp1 element. Exposure time used to record fluorescence images was constant in A and B, G and H and for I-L.