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Fig. 6 Notch signaling regulates sox9b expression in the intrahepatic and intrapancreatic ducts.
(A–D) Whole-mount in situ hybridization (ISH) showing sox9b expression in larvae obtained from a wild-type to sox9b heterozygote cross that were treated with DMSO control or 20 μM DAPT between 75 and 99 hpf. In DMSO-treated controls, sox9b was strongly expressed in the liver and pancreas at 99 hpf (A, Class I). In animals treated with 20 µM DAPT (B–D), some showed a slight reduction of sox9b expression in the liver (Li) and pancreas (Pa) (B, Class II), others only retained expression of sox9b in the liver (C, Class III), and the remaining ones did not exhibit any obvious expression of sox9b in either organ (D, Class IV). Dorsal views, anterior (A) to the top. (E) Percentages of larvae showing different classes of phenotypes. sox9b heterozygotes exhibited a more severe reduction in sox9b expression than wild-type upon DAPT treatment. The numbers of larvae analyzed are indicated at the bottom. (F–I) Larvae obtained from crossing Tg(hsp70l:Gal4);Tg(UAS:myc-Notch1a-intra) hemizygous and Tg(Tp1bglob:GFP);Tg(Tp1bglob:H2BmCherry) parents were heat-shocked at 80 hpf to induce myc-Notch1a-intra expression and fixed 26 hours later. (myc-Notch1a-intra)-overexpressing larvae were selected based on the increased expression of both Tg(Tp1bglob:GFP) and Tg(Tp1bglob:H2BmCherry), and their genotype was confirmed by anti-myc antibody labeling (data not shown). (F–G) in situ hybridization showed an increased expression of sox9b in the liver (Li) and pancreas (Pa) as well as in the HPD system (bracket) including the gallbladder (arrowhead) in (myc-Notch1a-intra)-overexpressing larvae (G) compared to (myc-Notch1a-intra)-negative siblings (F). 10 larvae of each genotype were analyzed. Dorsal views, anterior (A) to the top. (H–I) Confocal images of (myc-Notch1a-intra)-negative (H) and (myc-Notch1a-intra)-overexpressing (I) larvae showing elevated expression of Tg(Tp1bglob:GFP) and Tg(Tp1bglob:H2BmCherry) in the liver (Li), pancreas (Pa), gut (g) and gallbladder (gb) in (myc-Notch1a-intra)-overexpressing larvae after heat-shock. Dashed lines in (I) mark the pancreas. All images are projections of confocal z-stacks. Ventral views, anterior (A) to the top. Scale bars, 50 μm.