Journal: Genes & Development
Article Title: REST is a major negative regulator of endocrine differentiation during pancreas organogenesis
doi: 10.1101/gad.348501.121
Figure Lengend Snippet: 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) ( ; ) 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, χ 2 test.
Article Snippet: After three passages, we treated organoids with X5050 (Calbiochem) in human complete media for 48 h before RNA analysis.
Techniques: Transgenic Assay, Positive Control, Negative Control, Expressing
![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 <t>X5050,</t> 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) ( ; ) 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, χ 2 test.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_5321/pmc08415321/pmc08415321__1229f05.jpg)
