REST regulation of endocrine differentiation is conserved in zebrafish. (A) Embryos of Ins:mCherry/Gcga:GFP double-transgenic zebrafish line treated with 50 µM DAPT (Notch inhibitor used as positive control), 0.5 or 5 µM X5050, or vehicle (DMSO, negative control) from 3 dpf until 6 dpf. After drug treatment at 6 dpf, zebrafish pancreas was dissected, and the presence or absence of secondary islets was quantified. Arrows show representative secondary islets of double-transgenic zebrafish embryos (Ins:mCherry/Gcga:GFP; [blue] DAPI). The graph shows the percentage of zebrafish with detectable secondary islets in each condition. n = 20–25 fish per each condition. (B) An Ins:NTR-mCherry line was used to selectively ablate β cells upon treatment with 5 µM nifurpirinol (NFP) (Pisharath et al. 2007; Bergemann et al. 2018) in 3-dpf embryos; 24 h later after complete β-cell ablation of the principal islet, embryos were exposed to 5 µM ×5050 or vehicle, and β cells were analyzed 36 h later. Representative images of β-cell regeneration in Ins:NTR-mCherry embryos treated with vehicle (DMSO), with NFP only, or with NFP and X5050. (Red) Insulin, (blue) DAPI. The graph shows the percentage of pancreas showing >10 insulin-expressing cells in every condition. n = 32–36 fish per each condition. Scale bars, 200 µm. Error bars are SEM. (**) P < 0.01, (*) P < 0.05, χ2 test.
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